Anti-transthyretin antibodies

ABSTRACT

The invention provides antibodies that specifically bind transthyretin (TTR). The antibodies can be used for treating or effecting prophylaxis of diseases or disorders associated with TTR accumulation or accumulation of TTR deposits (e.g., TTR amyloidosis). The antibodies can also be used for diagnosing TTR amyloidosis and inhibiting or reducing aggregation of TTR, among other applications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 15/201,423filed Jul. 2, 2015, which is a continuation in part of U.S. applicationSer. No. 15/009,662 filed Jan. 28, 2016, which claims the benefit ofU.S. Provisional Application No. 62/109,002 filed Jan. 28, 2015 and U.S.Provisional Application No. 62/266,556 filed Dec. 11, 2015, each ofwhich is incorporated by reference in its entirety.

REFERENCE TO A SEQUENCE LISTING

This application includes an electronic sequence listing in a file named517666_SEQLST.TXT, created on Sep. 12, 2018, and containing 135,160bytes, which is hereby incorporated by reference in its entirety for allpurposes.

BACKGROUND

Several diseases are thought to be caused by the abnormal folding andaggregation of disease-specific proteins. These proteins can accumulateinto pathologically diagnostic accumulations, known as amyloids, whichare visualized by certain histologic stains. Amyloids are thought toelicit inflammatory responses and have multiple negative consequencesfor the involved tissues. In addition, smaller aggregates of abnormallyfolded protein may exist and exert cytotoxic effects.

Transthyretin (TTR) is one of the many proteins that are known tomisfold and aggregate (e.g., undergo amyloidogenesis).Transthyretin-related amyloidosis encompasses two forms of disease:familial disease arising from misfolding of a mutated or variant TTR,and a sporadic, non-genetic disease caused by misaggregation ofwild-type TTR. The process of TTR amyloidogenesis can cause pathology inthe nervous system and/or heart, as well as in other tissues.

SUMMARY OF THE CLAIMED INVENTION

The invention provides antibodies that bind to transthyretin andcomprise three heavy chain CDRs and three light chain CDRs substantiallyfrom antibody 14G8. Some antibodies comprise three Kabat heavy chainCDRs and three Kabat light chain CDRs of the antibody 14G8, except thatpositions H52 and L26 can each be N or S. Some antibodies comprise threeKabat heavy chain CDRs and three Kabat light chain CDRs of the antibody14G8. Optionally, CDR-H1 is a composite Kabat-Chothia CDR of theantibody 14G8. Some antibodies comprise three Kabat heavy chain CDRs ofthe antibody 9D5. Optionally, CDR-H1 is a composite Kabat-Chothia CDR ofthe antibody 9D5.

Some antibodies bind to the same epitope on transthyretin as 9D5 or 14G8and comprises three light chain CDRs and three heavy chain CDRs,wherein: (a) each CDR has at least 90% sequence identity to acorresponding CDR from the heavy and light chain variable regions of9D5; or (b) each CDR has at least 90% sequence identity to acorresponding CDR from the heavy and light chain variable regions of14G8, except that position L26 can be N or S. Some antibodies comprise:(a) the three heavy chain CDRs and the three light chain CDRs of 9D5; or(b) the three heavy chain CDRs and the three light chain CDRs of 14G8,except that position L26 can be N or S. In some antibodies, the threeheavy chain CDRs and the three light chain CDRs of 9D5 are SEQ ID NOS:13-15 and 24-26, respectively. In some antibodies, the three heavy chainCDRs and the three light chain CDRs of 14G8 are SEQ ID NOS: 67-69 and77-79, respectively, except that position L26 can be N or S.

Any of the above antibodies can be a monoclonal antibody. Any of theabove antibodies can be a chimeric, humanized, veneered, or humanantibody. Any of the above antibodies can have human IgG1 isotype, humanIgG2 isotype, or human IgG4 isotype.

The invention further provides humanized or chimeric antibodies of mouseantibody that specifically binds to transthyretin, wherein the mouseantibody is characterized by a mature heavy chain variable region of SEQID NO: 61 and a mature light chain variable region of SEQ ID NO: 70,except that position H52 can be S or N, position H69 can be F or I,position L26 can be S or N, and the Rat position L107 is optional.Optionally, the antibodies are a humanized or chimeric 9D5 or 14G8antibody that specifically binds to transthyretin, wherein 9D5 is amouse antibody characterized by a mature heavy chain variable region ofSEQ ID NO: 1 and a mature light chain variable region of SEQ ID NO: 16,and 14G8 is a mouse antibody characterized by a mature heavy chainvariable region of SEQ ID NO: 61 and a mature light chain variableregion of SEQ ID NO: 70. Optionally, the humanized antibodies comprise:(a) a humanized mature heavy chain variable region comprising the threeheavy chain CDRs of 9D5 and a humanized mature light chain variableregion comprising the three light chain CDRs of 9D5; or (b) a humanizedmature heavy chain variable region comprising the three heavy chain CDRsof 14G8 and a humanized mature light chain variable region comprisingthe three light chain CDRs of 14G8, except that position L26 can be N orS. Optionally, the three heavy chain CDRs and the three light chain CDRsof 9D5 are SEQ ID NOS: 13-15 and 24-26, respectively. Optionally, thethree heavy chain CDRs and the three light chain CDRs of 14G8 are SEQ IDNOS: 67-69 and 77-79, respectively, except that position L26 can be N orS. Optionally, any differences in CDRs of the mature heavy chainvariable region and mature light chain variable region from SEQ ID NOS:1 and 16, respectively, reside in positions H60-H65. Optionally, anydifferences in CDRs of the mature heavy chain variable region and maturelight chain variable region from SEQ ID NOS: 61 and 70, respectively,reside in positions H60-H65, except that position L26 can be N or S.

Optionally, the humanized antibody comprises a humanized mature heavychain variable region having an amino acid sequence at least 90%identical to any one of SEQ ID NOS: 5-12 and a humanized mature lightchain variable region having an amino acid sequence at least 90%identical to any one of SEQ ID NOS: 19-23, except that position H19 canbe R or K, position H40 can be A or T, position H44 can be G or R,position H49 can be S or A, position H77 can be S or T, position H82acan be N or S, position H83 can be R or K, position H84 can be A or S,and position H89 can be V or M. Optionally, at least one of thefollowing positions is occupied by the amino acid as specified: positionH42 is occupied by E, position H47 is occupied by L, position H69 isoccupied by F, position H82 is occupied by S, position H82b is occupiedby L, position H108 is occupied by L, position L8 is occupied by A,position L9 is occupied by P, position L18 is occupied by S, positionL19 is occupied by V, position L36 is occupied by F, position L39 isoccupied by R, position L60 is occupied by S, position L70 is occupiedby A, and position L74 is occupied by R. Optionally, positions H47, H69,and H82 are occupied by L, F, and S, respectively. Optionally, positionsH47, H69, H82, and H82b are occupied by L, F, S, and L, respectively.Optionally, positions H42, H47, and H108 are occupied by E, L, and L,respectively. Optionally, positions H69, H82, and H82b are occupied byF, S, and L, respectively. Optionally, positions H47 and H108 are eachoccupied by L. Optionally, positions H82 and H82b are occupied by S andL, respectively. Optionally, positions H42, H47, and H82b are occupiedby E, L, and L, respectively. Optionally, position L36 is occupied by F.Optionally, position L60 is occupied by S. Optionally, positions L8, L9,L19, L36, L39, L60, L70, and L74 are occupied by A, P, V, F, R, S, A,and R, respectively. Optionally, positions L8, L9, L18, L19, L36, L39,L60, L70, and L74 are occupied by A, P, S, V, F, R, S, A, and R,respectively.

Optionally, the humanized antibody comprises a mature heavy chainvariable region having an amino acid sequence at least 95% identical toany one of SEQ ID NOS: 5-12 and a mature light chain variable regionhaving an amino acid sequence at least 95% identical to any one of SEQID NOS: 19-23, except that position H19 can be R or K, position H40 canbe A or T, position H44 can be G or R, position H49 can be S or A,position H77 can be S or T, position H82a can be N or S, position H83can be R or K, position H84 can be A or S, and position H89 can be V orM. Optionally, the humanized antibody comprises a mature heavy chainvariable region having an amino acid sequence at least 98% identical toany one of SEQ ID NOS: 5-12 and a mature light chain variable regionhaving an amino acid sequence at least 98% identical to any one of SEQID NOS: 19-23, except that position H19 can be R or K, position H40 canbe A or T, position H44 can be G or R, position H49 can be S or A,position H77 can be S or T, position H82a can be N or S, position H83can be R or K, position H84 can be A or S, and position H89 can be V orM. Optionally, the mature heavy chain variable region has an amino acidsequence of any one of SEQ ID NO: 5-12 and the mature light chainvariable region has an amino acid sequence of any one of SEQ ID NO:19-23. In some antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO: 11 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO: 19.

Optionally, the humanized antibody comprises a humanized mature heavychain variable region having an amino acid sequence at least 90%identical to any one of SEQ ID NOS: 64-66 and a humanized mature lightchain variable region having an amino acid sequence at least 90%identical to any one of SEQ ID NOS: 74-76, except that position H82a canbe N or S, position H83 can be R or K, position H84 can be A or S,position H89 can be V or M, and position L18 can be S or P. Optionally,at least one of the following positions is occupied by the amino acid asspecified: position H1 is occupied by E, position H47 is occupied by L,and position L36 is occupied by F. Optionally, positions H1 and H47 areoccupied by E and L, respectively. Optionally, position L36 is occupiedby F. Optionally, at least one of the following positions is occupied bythe amino acid as specified: position H3 is occupied by K, position H105is occupied by T, position L8 is occupied by A, position L9 is occupiedby P, position L19 is occupied by V, position L26 is occupied by S,position L60 is occupied by S, and position L70 is occupied by A.Optionally, positions H3 and H105 are occupied by K and T, respectively.Optionally, positions L8, L9, L19, and L70 are occupied by A, P, V, andA, respectively. Optionally, positions L26 and L60 are each occupied byS.

Optionally, the humanized antibody comprises a mature heavy chainvariable region having an amino acid sequence at least 95% identical toany one of SEQ ID NOS: 64-66 and a mature light chain variable regionhaving an amino acid sequence at least 95% identical to any one of SEQID NOS: 74-76, except that position H82a can be N or S, position H83 canbe R or K, position H84 can be A or S, position H89 can be V or M, andposition L18 can be S or P. Optionally, the humanized antibody comprisesa mature heavy chain variable region having an amino acid sequence atleast 98% identical to any one of SEQ ID NOS: 64-66 and a mature lightchain variable region having an amino acid sequence at least 98%identical to any one of SEQ ID NOS: 74-76, except that position H82a canbe N or S, position H83 can be R or K, position H84 can be A or S,position H89 can be V or M, and position L18 can be S or P. Optionally,the mature heavy chain variable region has an amino acid sequence of anyone of SEQ ID NOS: 64-66, and the mature light chain variable region hasan amino acid sequence of any one of SEQ ID NOS: 74-76. In someantibodies, the mature heavy chain variable region has an amino acidsequence of SEQ ID NO: 65 and the mature light chain variable region hasan amino acid sequence of SEQ ID NO: 76.

Any of the above antibodies can be an intact antibody, a bindingfragment, a single-chain antibody, a Fab, or a Fab′2 fragment. In any ofthe above antibodies, the mature light chain variable region can befused to a light chain constant region and the mature heavy chainvariable region can be fused to a heavy chain constant region.Optionally, the heavy chain constant region is a mutant form of anatural human heavy chain constant region which has reduced binding to aFcγ receptor relative to the natural human heavy chain constant region.Optionally, the heavy chain constant region is of IgG1 isotype.Optionally, the mature heavy chain variable region is fused to a heavychain constant region having the sequence of SEQ ID NO: 103 and/or themature light chain variable region is fused to a light chain constantregion having the sequence of SEQ ID NO: 104 or 105.

The invention further provides pharmaceutical compositions comprisingany of the above antibodies and a pharmaceutically acceptable carrier.

The invention further provides nucleic acids encoding the heavy chainand/or light chain of any of the above antibodies, such as any one ofSEQ ID NOS: 40, 42, 44-56, 87, 89, 91-96, and 106-108.

The invention further provides a recombinant expression vectorcomprising a nucleic acid as described above, and a host celltransformed with the recombinant expression vector.

The invention further provides a method of humanizing an antibody, themethod comprising: (a) selecting one or more acceptor antibodies; (b)identifying the amino acid residues of the mouse antibody to beretained; (c) synthesizing a nucleic acid encoding a humanized heavychain comprising CDRs of the mouse antibody heavy chain and a nucleicacid encoding a humanized light chain comprising CDRs of the mouseantibody light chain; and (d) expressing the nucleic acids in a hostcell to produce a humanized antibody; wherein the mouse antibody is 9D5or 14G8, wherein 9D5 is characterized by a mature heavy chain variableregion of SEQ ID NO: 1 and a mature light chain variable region of SEQID NO: 16, and 14G8 is characterized by a mature heavy chain variableregion of SEQ ID NO: 61 and a mature light chain variable region of SEQID NO: 70.

The invention further provides a method of producing a humanized,chimeric, or veneered antibody, the method comprising: (a) culturingcells transformed with nucleic acids encoding the heavy and light chainsof the antibody, so that the cells secrete the antibody; and (b)purifying the antibody from cell culture media; wherein the antibody isa humanized, chimeric, or veneered form of 9D5 or 14G8.

The invention further provides a method of producing a cell lineproducing a humanized, chimeric, or veneered antibody, the methodcomprising: (a) introducing a vector encoding heavy and light chains ofan antibody and a selectable marker into cells; (b) propagating thecells under conditions to select for cells having increased copy numberof the vector; (c) isolating single cells from the selected cells; and(d) banking cells cloned from a single cell selected based on yield ofantibody; wherein the antibody is a humanized, chimeric, or veneeredform of 9D5 or 14G8. Optionally, the method further comprisespropagating the cells under selective conditions and screening for celllines naturally expressing and secreting at least 100 mg/L/10⁶ cells/24h.

The invention further provides a method of inhibiting or reducingaggregation of transthyretin in a subject having or at risk ofdeveloping a transthyretin-mediated amyloidosis, comprisingadministering to the subject an effective regime of any of the aboveantibodies, thereby inhibiting or reducing aggregation of transthyretinin the subject.

The invention further provides a method of inhibiting or reducingtransthyretin fibril formation in a subject having or at risk ofdeveloping a transthyretin-mediated amyloidosis, comprisingadministering to the subject an effective regime of any of the aboveantibodies, thereby inhibiting or reducing transthyretin accumulation inthe subject.

The invention further provides a method of reducing transthyretindeposits in a subject having or at risk of developing atransthyretin-mediated amyloidosis, comprising administering to thesubject an effective regime of any of the above antibodies, therebyreducing transthyretin deposits in the subject.

The invention further provides a method of clearing aggregatedtransthyretin in a subject having or at risk of developing atransthyretin-mediated amyloidosis, comprising administering to thesubject an effective regime of any of the above antibodies, therebyclearing aggregated transthyretin from the subject relative to a subjecthaving or at risk of developing a transthyretin-mediated amyloidosis whohas not received the antibody.

The invention further provides a method of stabilizing a non-toxicconformation of transthyretin in a subject having or at risk ofdeveloping a transthyretin-mediated amyloidosis, comprisingadministering to the subject an effective regime of any of the aboveantibodies, thereby stabilizing a non-toxic conformation oftransthyretin in the subject.

The invention further provides a method of treating or effectingprophylaxis of a transthyretin-mediated amyloidosis in a subject,comprising administering to the subject an effective regime of any ofthe above antibodies.

The invention further provides a method of delaying the onset of atransthyretin-mediated amyloidosis in a subject, comprisingadministering to the subject an effective regime of any of the aboveantibodies.

The invention further provides a method of diagnosing atransthyretin-mediated amyloidosis in a subject, comprising contacting abiological sample from the subject with an effective amount of any ofthe above antibodies. Optionally, the method further comprises detectingthe binding of antibody to transthyretin, wherein the presence of boundantibody indicates the subject has a transthyretin-mediated amyloidosis.Optionally, the method further comprises comparing binding of theantibody to the biological sample with binding of the antibody to acontrol sample, whereby increased binding of the antibody to thebiological sample relative to the control sample indicates the subjecthas a transthyretin-mediated amyloidosis. Optionally, the biologicalsample and the control sample comprise cells of the same tissue origin.Optionally, the biological sample and/or the control sample is blood,serum, plasma, or solid tissue. Optionally, the solid tissue is from theheart, peripheral nervous system, autonomic nervous system, kidneys,eyes, or gastrointestinal tract.

In any of the above methods, the transthyretin-mediated amyloidosis canbe associated with a condition selected from any of cardiomyopathy orhypertrophy, familial amyloid polyneuropathy, central nervous systemselective amyloidosis (CNSA), senile systemic amyloidosis, senilecardiac amyloidosis, spinal stenosis, osteoarthritis, rheumatoidarthritis, juvenile idiopathic arthritis, macular degeneration and aligament or tendon disorder.

The invention further provides a method of treating a subject having orat risk of any of cardiomyopathy or hypertrophy, familial amyloidpolyneuropathy, central nervous system selective amyloidosis (CNSA),senile systemic amyloidosis, senile cardiac amyloidosis, spinalstenosis, osteoarthritis, rheumatoid arthritis, juvenile idiopathicarthritis, macular degeneration and a ligament or tendon disorder, themethod comprising administering to the subject an effective regime ofthe antibody of any one of claims.

In any of the above methods, the transthyretin-mediated amyloidosis canoptionally be a familial transthyretin amyloidosis or a sporadictransthyretin amyloidosis. Optionally, the familial transthyretinamyloidosis is familial amyloid cardiomyopathy (FAC), familial amyloidpolyneuropathy (FAP), or central nervous system selective amyloidosis(CNSA). Optionally, the sporadic transthyretin amyloidosis is senilesystemic amyloidosis (SSA) or senile cardiac amyloidosis (SCA). In anyof the above methods, the transthyretin-mediated amyloidosis canoptionally be associated with amyloid accumulation in the heart,peripheral nervous system, autonomic nervous system, kidneys, eyes, orgastrointestinal tract of the subject.

The invention further provides a method of detecting the presence orabsence of transthyretin deposits in a subject, comprising contacting abiological sample from the subject suspected of comprising the amyloidaccumulation with an effective amount of any of the above antibodies.Optionally, the method further comprises detecting the binding ofantibody to transthyretin, wherein detection of bound antibody indicatesthe presence of transthyretin deposits. Optionally, the method furthercomprises comparing binding of the antibody to the biological samplewith binding of the antibody to a control sample, whereby increasedbinding of the antibody to the biological sample relative to the controlsample indicates the subject has a transthyretin-mediated amyloidosis.Optionally, the biological sample and the control sample comprise cellsof the same tissue origin. Optionally, the biological sample and/or thecontrol sample is blood, serum, plasma, or solid tissue. Optionally, thesolid tissue is from the heart, peripheral nervous system, autonomicnervous system, kidneys, eyes, or gastrointestinal tract.

The invention further provides a method of determining a level oftransthyretin deposits in a subject, comprising administering any of theabove antibodies and detecting the presence of bound antibody in thesubject. Optionally, the presence of bound antibody is determined bypositron emission tomography (PET).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A.1 & 1A.2 depict an alignment of heavy chain variable regions ofthe mouse 9D5 antibody, mouse model antibodies, human acceptorantibodies, and humanized versions of the 9D5 antibody. The CDRs asdefined by Kabat are enclosed in boxes, except that the first enclosedbox is a composite of the Chothia CDR-H1 and the Kabat CDR-H1, with theKabat CDR-H1 underlined and bolded.

FIG. 1B depicts an alignment of light chain variable regions of themouse 9D5 antibody, a mouse model antibody, a human acceptor antibody,and humanized versions of the 9D5 antibody. The CDRs as defined by Kabatare enclosed in boxes.

FIG. 2A depicts an alignment of heavy chain variable regions of themouse 14G8 antibody, a mouse model antibody, human acceptor antibodies,and humanized versions of the 14G8 antibody. The CDRs as defined byKabat are enclosed in boxes, except that the first enclosed box is acomposite of the Chothia CDR-H1 and the Kabat CDR-H1, with the KabatCDR-H1 underlined and bolded.

FIG. 2B depicts an alignment of light chain variable regions of themouse 14G8 antibody, a mouse model antibody, human acceptor antibodies,and humanized versions of the 14G8 antibody. The CDRs as defined byKabat are enclosed in boxes.

FIGS. 3A & 3B: FIG. 3A depicts binding curves of murine 5A1, 6C1, 9D5,and 14G8 antibodies to pH4-treated TTR. FIG. 3B depicts binding curvesof murine 5A1, 6C1, 9D5, and 14G8 antibodies to native TTR.

FIGS. 4A, 4B & 4C: FIG. 4A depicts the inhibition of TTR-Y78F fiberformation by mis-TTR antibodies. FIG. 4B depicts the inhibition ofTTR-V122I fiber formation by 14G8. FIG. 4C depicts the inhibition ofTTR-V122I fiber formation by a control antibody.

FIGS. 5A & 5B: FIG. 5A depicts a densitometry analysis of a Western blotanalysis of plasma samples from patients confirmed for V30M ATTR (Sample#11, #12, #15, #18, #19, #20) and samples from normal subjects (Sample#21, #22, #23, #24, #25, #27) using the 9D5 mis-TTR antibody. FIG. 5Bdepicts a densitometry analysis of a Western blot analysis of the samesamples using the 5A1 mis-TTR antibody.

FIG. 6 depicts a MesoScale Discovery (MSD) plate assay of plasma samplesfrom patients confirmed for V30M ATTR (Sample #11, #12, #15, #18, #19,#20) and samples from normal subjects (#21, #22, #23, #24, #25, #27)using the 6C1 antibody.

FIGS. 7A & 7B: FIG. 7A depicts the effect of antibody 14G8 on the uptakeof F87M/L110M TTR by THP-1 cells. FIG. 7B depicts the effect of each ofthe mis-TTR antibodies on the uptake of V30M TTR by THP-1 cells.

FIGS. 8A-D and FIGS. 8D.1-2: 14G8 binds to TTR-V122I fibril ends and tooligomeric aggregates as assessed using TEM and AFM. Immunogold labelingwith 14G8 was observed in TTR-V122I oligomer aggregates and fibril ends(FIG. 8A), whereas immunogold labeling with an anti-TTR pAb showedbinding along the lengths of TTR fibers and to oligomeric clusters (FIG.8B). IgG1 isotype control mAb did not show immunogold labeling (FIG.8C). TTR-V122I fibers, alone and in the presence of 14G8±6 nm colloidalgold-conjugated secondary antibody, were assessed using AFM. Goldlabeling was observed at fiber ends (FIG. 8D).

FIG. 9A.1-4 and FIG. 9B: Interaction of 14G8 with mature TTR-V122Ifibrils assessed using ITC fits to a 2-binding site model. ITC data andbinding isotherms for 14G8 binding to aggregated TTR variants arepresented in FIG. 8A. Binding was fit to a 2-binding site model withK_(D) values shown (FIG. 8B).

FIGS. 10A.1-2, 10B.1-2, 10C.1-2, 10D-F, and 10G.1-3 show 14G8immunolabeled TTR amyloid present between fibers of the nerve fascicle apatient with ATTR amyloidosis resulting from a TTR-V30M mutation. FIG.10A panels 1 and 2 show amyloid between fibers of the nerve fascicle,which overlapped with staining by Congo red (FIG. 8B panels 1 and 2) andthioflavin T (FIG. 10C panels 1 and 2), and immunolabeling by atotal-TTR antibody (FIG. 10D) in tissue derived from a patient with ATTRamyloidosis. No staining was seen with the use of 2 isotype controlantibodies (FIGS. 10E-F); however, axonal degeneration (lack of Schwanncell nuclei) in the areas laden with TTR amyloid deposits were alsoobserved (FIGS. 10E-F [red areas in 6E]). Peripheral nerves from ahealthy control were not labeled using either 14G8 or a total-TTRantibody (FIG. 10G panels 1-3).

FIGS. 11A.1-3, 11B.1-3, 11C.1-3, 11D.1-3, and 11E.1-4 shows antibody14G8 immunolabels TTR amyloid in the gastrointestinal tract derived froma patient with TTR-C30M amyloidosis. FIGS. 11A, B panels 1 showMeissner's plexus and glands in the esophagus, FIG. 11C panel 1 showsthe rich vasculature bed in the submucosa, FIG. 11D panel 1 shows themuscularis propria (MP) and muscularis mucosa 14G8-positive TTR amyloidoverlapped with Congo red fluorescent staining (FIGS. 11A-D panels 2).FIGS. 11A-D panels 3 show ATTR amyloidosis tissue stained with anisotype control mAb 14G8 immunoreactivity was absent in healthy controltissue (FIG. 11 panels 1-4).

FIGS. 12A-E show exemplary humanized 9D5 Vh designs, with backmutationsand other mutations based on selected human frameworks. The gray-shadedareas in the first column indicate the CDRs as defined by Chothia, andthe gray-shaded areas in the remaining columns indicate the CDRs asdefined by Kabat.

FIGS. 13A-D show exemplary humanized 9D5 Vk designs, with backmutationsand other mutations based on selected human frameworks. The gray-shadedareas in the first column indicate the CDRs as defined by Chothia, andthe gray-shaded areas in the remaining columns indicate the CDRs asdefined by Kabat.

FIGS. 14A-E show exemplary humanized 14G8 Vh designs, with backmutationsand other mutations based on selected human frameworks. The gray-shadedareas in the first column indicate the CDRs as defined by Chothia, andthe gray-shaded areas in the remaining columns indicate the CDRs asdefined by Kabat.

FIGS. 15A-D show exemplary humanized 14G8 Vk designs, with backmutationsand other mutations based on selected human frameworks. The gray-shadedareas in the first column indicate the CDRs as defined by Chothia, andthe gray-shaded areas in the remaining columns indicate the CDRs asdefined by Kabat.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 sets forth the amino acid sequence of the heavy chainvariable region of the mouse 9D5 antibody.

SEQ ID NO: 2 sets forth the amino acid sequence of the mouse heavy chainvariable region structure template 1SEQ_H.

SEQ ID NO: 3 sets forth the amino acid sequence of the heavy chainvariable acceptor ACC # BAC02114.

SEQ ID NO: 4 sets forth the amino acid sequence of the heavy chainvariable acceptor ACC # AAX82494.1.

SEQ ID NO: 5 sets forth the amino acid sequence of the heavy chainvariable region of the humanized 9D5 antibody version 1 (Hu9D5VHv1).

SEQ ID NO: 6 sets forth the amino acid sequence of the heavy chainvariable region of the humanized 9D5 antibody version 2 (Hu9D5VHv2).

SEQ ID NO: 7 sets forth the amino acid sequence of the heavy chainvariable region of the humanized 9D5 antibody version 2b (Hu9D5VHv2b).

SEQ ID NO: 8 sets forth the amino acid sequence of the heavy chainvariable region of the humanized 9D5 antibody version 3 (Hu9D5VHv3).

SEQ ID NO: 9 sets forth the amino acid sequence of the heavy chainvariable region of the humanized 9D5 antibody version 3b (Hu9D5VHv3b).

SEQ ID NO: 10 sets forth the amino acid sequence of the heavy chainvariable region of the humanized 9D5 antibody version 4 (Hu9D5VHv4).

SEQ ID NO: 11 sets forth the amino acid sequence of the heavy chainvariable region of the humanized 9D5 antibody version 4b (Hu9D5VHv4b).

SEQ ID NO: 12 sets forth the amino acid sequence of the heavy chainvariable region of the humanized 9D5 antibody version 5 (Hu9D5VHv5).

SEQ ID NO: 13 sets forth the amino acid sequence of Kabat CDR-H1 of themouse 9D5 antibody.

SEQ ID NO: 14 sets forth the amino acid sequence of Kabat CDR-H2 of themouse 9D5 antibody.

SEQ ID NO: 15 sets forth the amino acid sequence of Kabat CDR-H3 of themouse 9D5 antibody.

SEQ ID NO: 16 sets forth the amino acid sequence of the light chainvariable region of the mouse 9D5 antibody.

SEQ ID NO: 17 sets forth the amino acid sequence of the mouse lightchain variable region structure template 1MJU_L.

SEQ ID NO: 18 sets forth the amino acid sequence of the light chainvariable acceptor ACC # ABC66952.

SEQ ID NO: 19 sets forth the amino acid sequence of the light chainvariable region of the humanized 9D5 antibody version 1 (Hu9D5VLv1).

SEQ ID NO: 20 sets forth the amino acid sequence of the light chainvariable region of the humanized 9D5 antibody version 2 (Hu9D5VLv2).

SEQ ID NO: 21 sets forth the amino acid sequence of the light chainvariable region of the humanized 9D5 antibody version 3 (Hu9D5VLv3).

SEQ ID NO: 22 sets forth the amino acid sequence of the light chainvariable region of the humanized 9D5 antibody version 4 (Hu9D5VLv4).

SEQ ID NO: 23 sets forth the amino acid sequence of the light chainvariable region of the humanized 9D5 antibody version 5 (Hu9D5VLv5).

SEQ ID NO: 24 sets forth the amino acid sequence of Kabat CDR-L1 of themouse 9D5 antibody.

SEQ ID NO: 25 sets forth the amino acid sequence of Kabat CDR-L2 of themouse 9D5 antibody.

SEQ ID NO: 26 sets forth the amino acid sequence of Kabat CDR-L3 of themouse 9D5 antibody.

SEQ ID NO: 27 sets forth the amino acid sequence of humanized 9D5 heavychain version 1.

SEQ ID NO: 28 sets forth the amino acid sequence of humanized 9D5 heavychain version 2.

SEQ ID NO: 29 sets forth the amino acid sequence of humanized 9D5 heavychain version 2b.

SEQ ID NO: 30 sets forth the amino acid sequence of humanized 9D5 heavychain version 3.

SEQ ID NO: 31 sets forth the amino acid sequence of humanized 9D5 heavychain version 3b.

SEQ ID NO: 32 sets forth the amino acid sequence of humanized 9D5 heavychain version 4.

SEQ ID NO: 33 sets forth the amino acid sequence of humanized 9D5 heavychain version 4b.

SEQ ID NO: 34 sets forth the amino acid sequence humanized 9D5 heavychain version 5.

SEQ ID NO: 35 sets forth the amino acid sequence of humanized 9D5 lightchain version 1.

SEQ ID NO: 36 sets forth the amino acid sequence of humanized 9D5 lightchain version 2.

SEQ ID NO: 37 sets forth the amino acid sequence of humanized 9D5 lightchain version 3.

SEQ ID NO: 38 sets forth the amino acid sequence of humanized 9D5 lightchain version 4.

SEQ ID NO: 39 sets forth the amino acid sequence of humanized 9D5 lightchain version 5.

SEQ ID NO: 40 sets forth the nucleic acid sequence of the heavy chainvariable region of the mouse 9D5 antibody with signal peptide.

SEQ ID NO: 41 sets forth the amino acid sequence of the heavy chainvariable region of the mouse 9D5 antibody with signal peptide.

SEQ ID NO: 42 sets forth the nucleic acid sequence of the light chainvariable region of the mouse 9D5 antibody with signal peptide.

SEQ ID NO: 43 sets forth the amino acid sequence of the light chainvariable region of the mouse 9D5 antibody with signal peptide.

SEQ ID NO: 44 sets forth the nucleic acid sequence of the heavy chainvariable region of the humanized 9D5 antibody version 1 (Hu9D5VHv1).

SEQ ID NO: 45 sets forth the nucleic acid sequence of the heavy chainvariable region of the humanized 9D5 antibody version 2 (Hu9D5VHv2).

SEQ ID NO: 46 sets forth the nucleic acid sequence of the heavy chainvariable region of the humanized 9D5 antibody version 2b (Hu9D5VHv2b).

SEQ ID NO: 47 sets forth the nucleic acid sequence of the heavy chainvariable region of the humanized 9D5 antibody version 3 (Hu9D5VHv3).

SEQ ID NO: 48 sets forth the nucleic acid sequence of the heavy chainvariable region of the humanized 9D5 antibody version 3b (Hu9D5VHv3b).

SEQ ID NO: 49 sets forth the nucleic acid sequence of the heavy chainvariable region of the humanized 9D5 antibody version 4 (Hu9D5VHv4).

SEQ ID NO: 50 sets forth the nucleic acid sequence of the heavy chainvariable region of the humanized 9D5 antibody version 4b (Hu9D5VHv4b).

SEQ ID NO: 51 sets forth the nucleic acid sequence of the heavy chainvariable region of the humanized 9D5 antibody version 5 (Hu9D5VHv5).

SEQ ID NO: 52 sets forth the nucleic acid sequence of the light chainvariable region of the humanized 9D5 antibody version 1 (Hu9D5VLv1).

SEQ ID NO: 53 sets forth the nucleic acid sequence of the light chainvariable region of the humanized 9D5 antibody version 2 (Hu9D5VLv2).

SEQ ID NO: 54 sets forth the nucleic acid sequence of the light chainvariable region of the humanized 9D5 antibody version 3 (Hu9D5VLv3).

SEQ ID NO: 55 sets forth the nucleic acid sequence of the light chainvariable region of the humanized 9D5 antibody version 4 (Hu9D5VLv4).

SEQ ID NO: 56 sets forth the nucleic acid sequence of the light chainvariable region of the humanized 9D5 antibody version 5 (Hu9D5VLv5).

SEQ ID NO: 57 sets forth the amino acid sequence of the mouse 9D5 heavychain variable region signal peptide.

SEQ ID NO: 58 sets forth the nucleic acid sequence of the mouse 9D5heavy chain variable region signal peptide.

SEQ ID NO: 59 sets forth the amino acid sequence of the mouse 9D5 lightchain variable region signal peptide.

SEQ ID NO: 60 sets forth the nucleic acid sequence of the mouse 9D5light chain variable region signal peptide.

SEQ ID NO: 61 sets forth the amino acid sequence of the heavy chainvariable region of the mouse 14G8 antibody.

SEQ ID NO: 62 sets forth the amino acid sequence of the mouse heavychain variable region structure template 1MQK_H.

SEQ ID NO: 63 sets forth the amino acid sequence of the heavy chainvariable acceptor ACC # AAD30410.1.

SEQ ID NO: 64 sets forth the amino acid sequence of the heavy chainvariable region of the humanized 14G8 antibody version 1 (Hu14G8VHv1).

SEQ ID NO: 65 sets forth the amino acid sequence of the heavy chainvariable region of the humanized 14G8 antibody version 2 (Hu14G8VHv2).

SEQ ID NO: 66 sets forth the amino acid sequence of the heavy chainvariable region of the humanized 14G8 antibody version 3 (Hu14G8VHv3).

SEQ ID NO: 67 sets forth the amino acid sequence of Kabat CDR-H1 of themouse 14G8 antibody.

SEQ ID NO: 68 sets forth the amino acid sequence of Kabat CDR-H2 of themouse 14G8 antibody.

SEQ ID NO: 69 sets forth the amino acid sequence of Kabat CDR-H3 of themouse 14G8 antibody.

SEQ ID NO: 70 sets forth the amino acid sequence of the light chainvariable region of the mouse 14G8 antibody.

SEQ ID NO: 71 sets forth the amino acid sequence of the mouse lightchain variable region structure template 1MJU_L.

SEQ ID NO: 72 sets forth the amino acid sequence of the light chainvariable acceptor ACC # ABA71374.1.

SEQ ID NO: 73 sets forth the amino acid sequence of the light chainvariable acceptor ACC # ABC66952.1.

SEQ ID NO: 74 sets forth the amino acid sequence of the light chainvariable region of the humanized 14G8 antibody version 1 (Hu14G8VLv1).

SEQ ID NO: 75 sets forth the amino acid sequence of the light chainvariable region of the humanized 14G8 antibody version 2 (Hu14G8VLv2).

SEQ ID NO: 76 sets forth the amino acid sequence of the light chainvariable region of the humanized 14G8 antibody version 3 (Hu14G8VLv3).

SEQ ID NO: 77 sets forth the amino acid sequence of Kabat CDR-L1 of themouse 14G8 antibody.

SEQ ID NO: 78 sets forth the amino acid sequence of Kabat CDR-L2 of themouse 14G8 antibody.

SEQ ID NO: 79 sets forth the amino acid sequence of Kabat CDR-L3 of themouse 14G8 antibody.

SEQ ID NO: 80 sets forth the amino acid sequence of Kabat CDR-L1 of thehumanized 14G8 antibody version 3 (Hu14G8VLv3).

SEQ ID NO: 81 sets forth the amino acid sequence of humanized 14G8 heavychain version 1.

SEQ ID NO: 82 sets forth the amino acid sequence of humanized 14G8 heavychain version 2.

SEQ ID NO: 83 sets forth the amino acid sequence of humanized 14G8 heavychain version 3.

SEQ ID NO: 84 sets forth the amino acid sequence of humanized 14G8 lightchain version 1.

SEQ ID NO: 85 sets forth the amino acid sequence of humanized 14G8 lightchain version 2.

SEQ ID NO: 86 sets forth the amino acid sequence of humanized 14G8 lightchain version 3.

SEQ ID NO: 87 sets forth the nucleic acid sequence of the heavy chainvariable region of the mouse 14G8 antibody with signal peptide.

SEQ ID NO: 88 sets forth the amino acid sequence of the heavy chainvariable region of the mouse 14G8 antibody with signal peptide.

SEQ ID NO: 89 sets forth the nucleic acid sequence of the light chainvariable region of the mouse 14G8 antibody with signal peptide.

SEQ ID NO: 90 sets forth the amino acid sequence of the light chainvariable region of the mouse 14G8 antibody with signal peptide.

SEQ ID NO: 91 sets forth the nucleic acid sequence of the heavy chainvariable region of the humanized 14G8 antibody version 1 (Hu14G8VHv1).

SEQ ID NO: 92 sets forth the nucleic acid sequence of the heavy chainvariable region of the humanized 14G8 antibody version 2 (Hu14G8VHv2).

SEQ ID NO: 93 sets forth the nucleic acid sequence of the heavy chainvariable region of the humanized 14G8 antibody version 3 (Hu14G8VHv3).

SEQ ID NO: 94 sets forth the nucleic acid sequence of the light chainvariable region of the humanized 14G8 antibody version 1 (Hu14G8VLv1).

SEQ ID NO: 95 sets forth the nucleic acid sequence of the light chainvariable region of the humanized 14G8 antibody version 2 (Hu14G8VLv2).

SEQ ID NO: 96 sets forth the nucleic acid sequence of the light chainvariable region of the humanized 14G8 antibody version 3 (Hu14G8VLv3).

SEQ ID NO: 97 sets forth the amino acid sequence of the mouse 14G8 heavychain variable region signal peptide.

SEQ ID NO: 98 sets forth the nucleic acid sequence of the mouse 14G8heavy chain variable region signal peptide.

SEQ ID NO: 99 sets forth the amino acid sequence of the mouse 14G8 lightchain variable region signal peptide.

SEQ ID NO: 100 sets forth the nucleic acid sequence of the mouse 14G8light chain variable region signal peptide.

SEQ ID NO: 101 sets forth the amino acid sequence of an exemplary humanIgG1 heavy chain constant region.

SEQ ID NO: 102 sets forth the amino acid sequence of an exemplary humanIgG1 heavy chain constant region of the IgG1 G1m3 allotype.

SEQ ID NO: 103 sets forth the amino acid sequence of an exemplary humanIgG1 heavy chain constant region of the IgG1 G1m3 allotype.

SEQ ID NO: 104 sets forth the amino acid sequence of an exemplary humankappa light chain constant region having an N-terminal arginine.

SEQ ID NO: 105 sets forth the amino acid sequence of an exemplary humankappa light chain constant region without an N-terminal arginine.

SEQ ID NO: 106 sets forth the nucleic acid sequence of an exemplaryheavy chain constant region of the G1m3 allotype.

SEQ ID NO: 107 sets forth the nucleic acid sequence of an exemplarylight chain constant region having an N-terminal arginine.

SEQ ID NO: 108 sets forth the nucleic acid sequence of an exemplarylight chain constant region without an N-terminal arginine.

SEQ ID NO: 109 sets forth the amino acid sequence of human transthyretinset forth in accession number P02766.1 (UniProt).

SEQ ID NO: 110 sets forth the amino acid sequence of human transthyretinset forth in accession number AAB35639.1 (GenBank).

SEQ ID NO: 111 sets forth the amino acid sequence of human transthyretinset forth in accession number AAB35640.1 (GenBank).

SEQ ID NO: 112 sets forth the amino acid sequence of human transthyretinset forth in accession number and ABI63351.1 (GenBank).

SEQ ID NO: 113 sets forth the amino acid sequence of residues 89-97 ofhuman transthyretin.

SEQ ID NO: 114 sets forth the amino acid sequence of a potentialtransthyretin immunogen.

SEQ ID NO: 115 sets forth the amino acid sequence of a potentialtransthyretin immunogen.

SEQ ID NO: 116 sets forth the amino acid sequence of a potentialtransthyretin immunogen.

SEQ ID NO: 117 sets forth the amino acid sequence of compositeChothia-Kabat CDR-H1 of the mouse 9D5 antibody.

SEQ ID NO: 118 sets forth the amino acid sequence of compositeChothia-Kabat CDR-H1 of the mouse 14G8 antibody.

DEFINITIONS

Monoclonal antibodies or other biological entities are typicallyprovided in isolated form. This means that an antibody or otherbiologically entity is typically at least 50% w/w pure of interferingproteins and other contaminants arising from its production orpurification but does not exclude the possibility that the monoclonalantibody is combined with an excess of pharmaceutically acceptablecarrier(s) or other vehicle intended to facilitate its use. Sometimesmonoclonal antibodies are at least 60%, 70%, 80%, 90%, 95% or 99% w/wpure of interfering proteins and contaminants from production orpurification. Often an isolated monoclonal antibody or other biologicalentity is the predominant macromolecular species remaining after itspurification.

Specific binding of an antibody to its target antigen means an affinityof at least 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹. Specific binding isdetectably higher in magnitude and distinguishable from non-specificbinding occurring to at least one unrelated target. Specific binding canbe the result of formation of bonds between particular functional groupsor particular spatial fit (e.g., lock and key type) whereas nonspecificbinding is usually the result of van der Waals forces. Specific bindingdoes not however necessarily imply that an antibody binds one and onlyone target.

The basic antibody structural unit is a tetramer of subunits. Eachtetramer includes two identical pairs of polypeptide chains, each pairhaving one “light” (about 25 kDa) and one “heavy” chain (about 50-70kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. This variable region is initially expressed linkedto a cleavable signal peptide. The variable region without the signalpeptide is sometimes referred to as a mature variable region. Thus, forexample, a light chain mature variable region means a light chainvariable region without the light chain signal peptide. Thecarboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 or more amino acids. See generally,Fundamental Immunology, Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989,Ch. 7 (incorporated by reference in its entirety for all purposes).

An immunoglobulin light or heavy chain variable region (also referred toherein as a “light chain variable domain” (“VL domain”) or “heavy chainvariable domain” (“VH domain”), respectively) consists of a “framework”region interrupted by three “complementarity determining regions” or“CDRs.” The framework regions serve to align the CDRs for specificbinding to an epitope of an antigen. The CDRs include the amino acidresidues of an antibody that are primarily responsible for antigenbinding. From amino-terminus to carboxyl-terminus, both VL and VHdomains comprise the following framework (FR) and CDR regions: FR1,CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDRs 1, 2, and 3 of a VL domain arealso referred to herein, respectively, as CDR-L1, CDR-L2, and CDR-L3;CDRs 1, 2, and 3 of a VH domain are also referred to herein,respectively, as CDR-H1, CDR-H2, and CDR-H3.

The assignment of amino acids to each VL and VH domain is in accordancewith any conventional definition of CDRs. Conventional definitionsinclude, the Kabat definition (Kabat, Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.,1987 and 1991), The Chothia definition (Chothia & Lesk, J. Mol. Biol.196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); acomposite of Chothia Kabat CDR in which CDR-H1 is a composite of Chothiaand Kabat CDRs; the AbM definition used by Oxford Molecular's antibodymodelling software; and, the contact definition of Martin et al(bioinfo.org.uk/abs) (see Table 1). Kabat provides a widely usednumbering convention (Kabat numbering) in which corresponding residuesbetween different heavy chains or between different light chains areassigned the same number. When an antibody is said to comprise CDRs by acertain definition of CDRs (e.g., Kabat) that definition specifies theminimum number of CDR residues present in the antibody (i.e., the KabatCDRs). It does not exclude that other residues falling within anotherconventional CDR definition but outside the specified definition arealso present. For example, an antibody comprising CDRs defined by Kabatincludes among other possibilities, an antibody in which the CDRscontain Kabat CDR residues and no other CDR residues, and an antibody inwhich CDR H1 is a composite Chothia-Kabat CDR H1 and other CDRs containKabat CDR residues and no additional CDR residues based on otherdefinitions.

TABLE 1 Conventional Definitions of CDRs Using Kabat Numbering Compositeof Chothia Loop Kabat Chothia & Kabat AbM Contact L1 L24-L34 L24-L34L24-L34 L24-L34 L30-L36 L2 L50-L56 L50-L56 L50-L56 L50-L56 L46-L55 L3L89-L97 L89-L97 L89-L97 L89-L97 L89-L96 H1 H31-H35B H26-H32 . . . H34*H26-H35B* H26-H35B H30-H35B H2 H50-H65 H52-H56 H50-H65 H50-H58 H47-H58H3 H95-H102 H95-H102 H95-H102 H95-H102 H93-H101 *CDR-H1 by Chothia canend at H32, H33, or H34 (depending on the length of the loop). This isbecause the Kabat numbering scheme places insertions of extra residuesat 35A and 35B, whereas Chothia numbering places them at 31A and 31B. Ifneither H35A nor H35B (Kabat numbering) is present, the Chothia CDR-H1loop ends at H32. If only H35A is present, it ends at H33. If both H35Aand H35B are present, it ends at H34.

The term “antibody” includes intact antibodies and binding fragmentsthereof. Typically, fragments compete with the intact antibody fromwhich they were derived for specific binding to the target includingseparate heavy chains, light chains Fab, Fab′, F(ab′)₂, F(ab)c, Dabs,nanobodies, and Fv. Fragments can be produced by recombinant DNAtechniques, or by enzymatic or chemical separation of intactimmunoglobulins. The term “antibody” also includes a bispecific antibodyand/or a humanized antibody. A bispecific or bifunctional antibody is anartificial hybrid antibody having two different heavy/light chain pairsand two different binding sites (see, e.g., Songsivilai and Lachmann,Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol.,148:1547-53 (1992)). In some bispecific antibodies, the two differentheavy/light chain pairs include a humanized 9D5 heavy chain/light chainpair and a heavy chain/light chain pair specific for a different epitopeon transthyretin than that bound by 9D5. In some bispecific antibodies,the two different heavy/light chain pairs include a humanized 14G8 heavychain/light chain pair and a heavy chain/light chain pair specific for adifferent epitope on transthyretin than that bound by 14G8.

In some bispecific antibodies, one heavy chain/light chain pair is ahumanized 9D5 or 14G8 antibody as further disclosed below and the otherheavy chain/light chain pair is from an antibody that binds to areceptor expressed on the blood brain barrier, such as an insulinreceptor, an insulin-like growth factor (IGF) receptor, a leptinreceptor, or a lipoprotein receptor, or a transferrin receptor (Fridenet al., Proc. Natl. Acad. Sci. USA 88:4771-4775, 1991; Friden et al.,Science 259:373-377, 1993). Such a bispecific antibody can betransferred cross the blood brain barrier by receptor-mediatedtranscytosis. Brain uptake of the bispecific antibody can be furtherenhanced by engineering the bi-specific antibody to reduce its affinityto the blood brain barrier receptor. Reduced affinity for the receptorresulted in a broader distributioin in the brain (see, e.g., Atwal etal., Sci. Trans. Med. 3, 84ra43, 2011; Yu et al., Sci. Trans. Med. 3,84ra44, 2011).

Exemplary bispecific antibodies can also be: (1) a dual-variable-domainantibody (DVD-Ig), where each light chain and heavy chain contains twovariable domains in tandem through a short peptide linkage (Wu et al.,Generation and Characterization of a Dual Variable Domain Immunoglobulin(DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg(2010)); (2) a Tandab, which is a fusion of two single chain diabodiesresulting in a tetravalent bispecific antibody that has two bindingsites for each of the target antigens; (3) a flexibody, which is acombination of scFvs with a diabody resulting in a multivalent molecule;(4) a so-called “dock and lock” molecule, based on the “dimerization anddocking domain” in Protein Kinase A, which, when applied to Fabs, canyield a trivalent bispecific binding protein consisting of two identicalFab fragments linked to a different Fab fragment; or (5) a so-calledScorpion molecule, comprising, e.g., two scFvs fused to both termini ofa human Fc-region. Examples of platforms useful for preparing bispecificantibodies include Bi (Micromet), DART (MacroGenics), Fcab and Mab2(F-star), Fc-engineered IgG1 (Xencor) or DuoBody (based on Fab armexchange, Genmab).

The term “epitope” refers to a site on an antigen to which an antibodybinds. An epitope can be formed from contiguous amino acids ornoncontiguous amino acids juxtaposed by tertiary folding of one or moreproteins. Epitopes formed from contiguous amino acids (also known aslinear epitopes) are typically retained on exposure to denaturingsolvents whereas epitopes formed by tertiary folding (also known asconformational epitopes) are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed. (1996). The epitope can be linear, such as anepitope of, for example, 2-5, 3-5, 3-9, or 5-9 contiguous amino acidsfrom SEQ ID NO: 109. The epitope can also be a conformational epitopeincluding, for example, two or more non-contiguous segments of aminoacids within residues 89-97 of SEQ ID NO: 109.

If an antibody is said to bind to an epitope within amino acids 89-97 oftransthyretin (TTR), for example, what is meant is that the epitope iswithin the recited range of amino acids including those defining theouter-limits of the range. It does not necessarily mean that every aminoacid within the range constitutes part of the epitope. Thus, forexample, an epitope within amino acids 89-97 of TTR may consist of aminoacids 89-97, 89-96, 90-96, 91-96, 92-96, 93-96, 94-96, 89-96, 89-95,89-94, 89-93, 89-92 or 89-93, among other linear segments of SEQ ID NO:113, or in the case of conformational epitopes, non-contiguous segmentsof amino acids of SEQ ID NO: 113.

Antibodies that recognize the same or overlapping epitopes can beidentified in a simple immunoassay showing the ability of one antibodyto compete with the binding of another antibody to a target antigen. Theepitope of an antibody can also be defined X-ray crystallography of theantibody bound to its antigen to identify contact residues.Alternatively, two antibodies have the same epitope if all amino acidmutations in the antigen that reduce or eliminate binding of oneantibody reduce or eliminate binding of the other. Two antibodies haveoverlapping epitopes if some amino acid mutations that reduce oreliminate binding of one antibody reduce or eliminate binding of theother.

Competition between antibodies is determined by an assay in which anantibody under test inhibits specific binding of a reference antibody toa common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495,1990). A test antibody competes with a reference antibody if an excessof a test antibody (e.g., at least 2×, 5×, 10×, 20× or 100×) inhibitsbinding of the reference antibody by at least 50% as measured in acompetitive binding assay. Some test antibodies inhibit binding of thereferences antibody by at least 75%, 90% or 99%. Antibodies identifiedby competition assay (competing antibodies) include antibodies bindingto the same epitope as the reference antibody and antibodies binding toan adjacent epitope sufficiently proximal to the epitope bound by thereference antibody for steric hindrance to occur.

The term “native” with respect to the structure transthyretin (TTR)refers to the normal folded structure of TTR in its properly functioningstate (i.e., a TTR tetramer). As TTR is a tetramer in its nativelyfolded form, non-native forms of TTR include, for example, misfolded TTRtetramers, TTR monomers, aggregated forms of TTR, and fibril forms ofTTR. Non-native forms of TTR can include molecules comprising wild-typeTTR amino acid sequences or mutations.

The term “misfolded” with respect to TTR refers to the secondary andtertiary structure of a TTR polypeptide monomer or multimer, andindicates that the polypeptide has adopted a conformation that is notnormal for that protein in its properly functioning state. Although TTRmisfolding can be caused by mutations in the protein (e.g., deletion,substitution, or addition), wild-type TTR proteins can also be misfoldedin diseases, exposing specific epitopes.

The term “pharmaceutically acceptable” means that the carrier, diluent,excipient, or auxiliary is compatible with the other ingredients of theformulation and not substantially deleterious to the recipient thereof.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

An individual is at increased risk of a disease if the subject has atleast one known risk-factor (e.g., genetic, biochemical, family history,and situational exposure) placing individuals with that risk factor at astatistically significant greater risk of developing the disease thanindividuals without the risk factor.

The term “biological sample” refers to a sample of biological materialwithin or obtainable from a biological source, for example a human ormammalian subject. Such samples can be organs, organelles, tissues,sections of tissues, bodily fluids, peripheral blood, blood plasma,blood serum, cells, molecules such as proteins and peptides, and anyparts or combinations derived therefrom. The term biological sample canalso encompass any material derived by processing the sample. Derivedmaterial can include cells or their progeny. Processing of thebiological sample may involve one or more of filtration, distillation,extraction, concentration, fixation, inactivation of interferingcomponents, and the like.

The term “control sample” refers to a biological sample not known orsuspected to include monomeric, misfolded, aggregated, or fibril formsof transthyretin (TTR), such as in TTR amyloid deposits. Control samplescan be obtained from individuals not afflicted with a TTR amyloidosis ora specifically chosen type of TTR amyloidosis. Alternatively, controlsamples can be obtained from patients afflicted with TTR amyloidosis ora specifically chosen type of TTR amyloidosis. Such samples can beobtained at the same time as a biological sample thought to comprise theTTR amyloidosis or on a different occasion. A biological sample and acontrol sample can both be obtained from the same tissue (e.g., a tissuesection containing both TTR amyloid deposits and surrounding normaltissue). Preferably, control samples consist essentially or entirely oftissue free of TTR amyloid deposits and can be used in comparison to abiological sample thought to comprise TTR amyloid deposits. Preferably,the tissue in the control sample is the same type as the tissue in thebiological sample (e.g., cardiomyocytes in the heart).

The term “disease” refers to any abnormal condition that impairsphysiological function. The term is used broadly to encompass anydisorder, illness, abnormality, pathology, sickness, condition, orsyndrome in which physiological function is impaired, irrespective ofthe nature of the etiology.

The term “symptom” refers to a subjective evidence of a disease, such asaltered gait, as perceived by the subject. A “sign” refers to objectiveevidence of a disease as observed by a physician.

For purposes of classifying amino acids substitutions as conservative ornonconservative, amino acids are grouped as follows: Group I(hydrophobic side chains): met, ala, val, leu, ile; Group II (neutralhydrophilic side chains): cys, ser, thr; Group III (acidic side chains):asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V(residues influencing chain orientation): gly, pro; and Group VI(aromatic side chains): trp, tyr, phe. Conservative substitutionsinvolve substitutions between amino acids in the same class.Nonconservative substitutions constitute exchanging a member of one ofthese classes for a member of another.

Percentage sequence identities are determined with antibody sequencesmaximally aligned by the Kabat numbering convention. After alignment, ifa subject antibody region (e.g., the entire mature variable region of aheavy or light chain) is being compared with the same region of areference antibody, the percentage sequence identity between the subjectand reference antibody regions is the number of positions occupied bythe same amino acid in both the subject and reference antibody regiondivided by the total number of aligned positions of the two regions,with gaps not counted, multiplied by 100 to convert to percentage.

Compositions or methods “comprising” or “including” one or more recitedelements may include other elements not specifically recited. Forexample, a composition that “comprises” or “includes” an antibody maycontain the antibody alone or in combination with other ingredients.

Designation of a range of values includes all integers within ordefining the range, and all subranges defined by integers within therange.

Unless otherwise apparent from the context, the term “about” encompassesvalues within a standard margin of error of measurement (e.g., SEM) of astated value.

Statistical significance means p≤0.05.

The singular forms of the articles “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” can include a pluralityof compounds, including mixtures thereof.

DETAILED DESCRIPTION I. General

The invention provides antibodies that specifically bind to residues89-97 of transthyretin (TTR). The antibodies have the capacity to bindto monomeric, misfolded, aggregated, or fibril forms of TTR. Theantibodies can be used for treating or effecting prophylaxis of diseasesor disorders associated with TTR accumulation or accumulation of TTRdeposits (e.g., TTR amyloidosis). The antibodies can also be used fordiagnosing TTR amyloidosis and inhibiting or reducing aggregation ofTTR, among other applications.

II. Target Molecules

Transthyretin (TTR) is a 127-amino acid, 55 kDa serum and cerebrospinalfluid transport protein primarily synthesized by the liver. It has alsobeen referred to as prealbumin, thyroxine binding prealbumin, ATTR, andTBPA. In its native state, TTR exists as a tetramer. In homozygotes, thetetramers comprise identical 127-amino-acid beta-sheet-rich subunits. Inheterozygotes, the TTR tetramers are made up of variant and/or wild-typesubunits, typically combined in a statistical fashion.

The established function of TTR in the blood is to transportholo-retinol binding protein. Although TTR is the major carrier ofthyroxine (T₄) in the blood of rodents, utilizing binding sites that areorthogonal to those used for holo-retinol binding protein, the T₄binding sites are effectively unoccupied in humans.

TTR is one of at least thirty different human proteins whoseextracellular misfolding and/or misassembly (amyloidogenesis) into aspectrum of aggregate structures is thought to cause degenerativediseases referred to as amyloid diseases. TTR undergoes conformationalchanges in order to become amyloidogenic. Partial unfolding exposesstretches of largely uncharged hydrophobic residues in an extendedconformation that efficiently misassemble into largely unstructuredspherical aggregates that ultimately undergo conformation conversioninto cross-beta sheet amyloid structures.

Unless otherwise apparent from context, reference to transthyretin (TTR)or its fragments or domains includes the natural human amino acidsequences including isoforms, mutants, and allelic variants thereof.Exemplary TTR polypeptide sequences are designated by Accession NumbersP02766.1 (UniProt), AAB35639.1 (GenBank), AAB35640.1 (GenBank), andABI63351.1 (GenBank) (SEQ ID NOS: 109-112, respectively). Residues arenumbered according to Swiss Prot P02766.1, with the first amino acid ofthe mature protein (i.e., not including the 20 amino acid signalsequence) designated residue 1. In any other TTR protein, residues arenumbered according to the corresponding residues in P02766.1 on maximumalignment.

III. Transthyretin Amyloidosis

Transthyretin (TTR) amyloidosis is a systemic disorder characterized bypathogenic, misfolded TTR and the extracellular deposition of amyloidfibrils composed of TTR. TTR amyloidosis is generally caused bydestabilization of the native TTR tetramer form (due to environmental orgenetic conditions), leading to dissociation, misfolding, andaggregation of TTR into amyloid fibrils that accumulate in variousorgans and tissues, causing progressive dysfunction. See, e.g., Almeidaand Saraiva, FEBS Letters 586:2891-2896 (2012); Ando et al., OrphanetJournal of Rare Diseases 8:31 (2013).

In humans, both wild-type TTR tetramers and mixed tetramers comprised ofmutant and wild-type subunits can dissociate, misfold, and aggregate,with the process of amyloidogenesis leading to the degeneration ofpost-mitotic tissue. Thus, TTR amyloidoses encompass diseases caused bypathogenic misfolded TTR resulting from mutations in TTR or resultingfrom non-mutated, misfolded TTR.

For example, senile systemic amyloidosis (SSA) and senile cardiacamyloidosis (SCA) are age-related types of amyloidosis that result fromthe deposition of wild-type TTR amyloid outside and within thecardiomyocytes of the heart. TTR amyloidosis is also the most commonform of hereditary (familial) amyloidosis, which is caused by mutationsthat destabilize the TTR protein. The TTR amyloidoses associated withpoint mutations in the TTR gene include familial amyloid polyneuropathy(FAP), familial amyloid cardiomyopathy (FAC), and the rare centralnervous system selective amyloidosis (CNSA). Patients with hereditary(familial) TTR amyloidosis are almost always heterozygotes, meaning thatthe TTR tetramers are composed of mutant and/or wild-type TTR subunits,generally statistically distributed. Hereditary (familial) versions ofTTR amyloidosis are generally autosomal dominant and are typicallyearlier onset than the sporadic diseases (SSA and SCA).

There are over 100 mutations in the gene encoding TTR that have beenimplicated in the autosomal dominant disorders FAP and FAC. See, e.g.,US 2014/0056904; Saraiva, Hum. Mutat. 17(6):493-503 (2001); Damas andSaraiva, J. Struct. Biol. 130:290-299; Dwulet and Benson, Biochem.Biophys. Res. Commun. 114:657-662 (1983). These amyloid-causingmutations are distributed throughout the entire molecule of TTR.Generally, the more destabilizing the mutant subunits are to the TTRtetramer structure, the earlier the onset of amyloid disease. Thepathogenic potential of a TTR variant is generally determined by acombination of its instability and its cellular secretion efficiency.The initial pathology caused by some TTR variants comes from theirselective destruction of cardiac tissue, whereas that from other TTRvariants comes from compromising the peripheral and autonomic nervoussystem. The tissue damage caused by TTR amyloidogenesis appear to stemlargely from the toxicity of small, diffusible TTR aggregates, althoughaccumulation of extracellular amyloid may contribute and almostcertainly compromises organ structure in the late stages of the TTRamyloidosis.

TTR amyloidosis presents in many different forms, with considerablephenotypic variation across individuals and geographic locations. Forexample, TTR amyloidosis can present as a progressive, axonal sensoryautonomic and motor neuropathy. TTR amyloidosis can also present as aninfiltrative cardiomyopathy.

The age at onset of disease-related symptoms varies between the secondand ninth decades of life, with great variations across differentpopulations. The multisystem involvement of TTR amyloidosis is a clue toits diagnosis. For example, TTR amyloidosis diagnosis is considered whenone or several of the following are present: (1) family history ofneuropathic disease, especially associated with heart failure; (2)neuropathic pain or progressive sensory disturbances of unknownetiology; (3) carpal tunnel syndrome without obvious cause, particularlyif it is bilateral and requires surgical release; (4) gastrointestinalmotility disturbances or autonomic nerve dysfunction of unknown etiology(e.g., erectile dysfunction, orthostatic hypotension, neurogenicgladder); (5) cardiac disease characterized by thickened ventricularwalls in the absence of hypertension; (6) advanced atrio-ventricularblock of unknown origin, particularly when accompanied by a thickenedheart; and (6) vitreous body inclusions of the cotton-wool type. SeeAndo et al., Orphanet Journal of Rare Diseases 8:31 (2013). Othersymptoms can include, for example, polyneuropathy, sensory loss, pain,weakness in lower limbs, dyshidrosis, diarrhea, constipation, weightloss, and urinary incontinence/retention.

Diagnosis of TTR amyloidosis typically relies on target organ biopsies,followed by histological staining of the excised tissue with theamyloid-specific dye, Congo red. If a positive test for amyloid isobserved, immunohistochemical staining for TTR is subsequently performedto ensure that the precursor protein responsible for amyloid formationis indeed TTR. For familial forms of the diseases, demonstration of amutation in the gene encoding TTR is then needed before diagnosis can bemade. This can be accomplished, for example, through isoelectricfocusing electrophoresis, polymerase chain reaction, or laserdissection/liquid chromatography-tandem mass spectrometry. See, e.g., US2014/0056904; Ruberg and Berk, Circulation 126:1286-1300 (2012); Ando etal., Orphanet Journal of Rare Diseases 8:31 (2013).

IV. Antibodies

A. Binding Specificity and Functional Properties

The invention provides monoclonal antibodies binding to transthyretin(TTR) protein, more specifically, to epitopes within amino acid residues89-97 (SEQ ID NO: 113) of TTR. Such epitopes are buried in the nativeTTR tetramer and exposed in monomeric, misfolded, aggregated, or fibrilforms of TTR.

Antibodies designated 9D5 and 14G8 are two such exemplary mouseantibodies. Unless otherwise apparent from context, reference to 9D5 or14G8 should be understood as referring to any of the mouse, chimeric,veneered, and humanized forms of these antibodies. These antibodiesspecifically bind within amino acid residues 89-97 (SEQ ID NO: 113) ofTTR. These antibodies are further characterized by their ability to bindto monomeric, misfolded, aggregated, or fibril forms of TTR but not tonative tetrameric forms of TTR. In addition, these antibodies arecharacterized by their immunoreactivity on TTR-mediated amyloidosiscardiac tissue but not on healthy cardiac tissue. Ability to bind tospecific proteins or fragments thereof may be demonstrated usingexemplary assay formats provided in the examples.

Some antibodies bind to the same or overlapping epitope as an antibodydesignated 9D5 or 14G8. The sequences of the heavy and light chainmature variable regions of these antibodies are designated SEQ ID NOS: 1and 16 (9D5), and 61 and 70 (14G8), respectively. Other antibodieshaving such a binding specificity can be produced by immunizing micewith TTR, or a portion thereof including the desired epitope (e.g., SEQID NO: 113), and screening resulting antibodies for binding to monomericTTR or a peptide comprising SEQ ID NO: 113, optionally in competitionwith an antibody having the variable regions of mouse 9D5 or 14G8 (IgG1kappa). Fragments of TTR including the desired epitope can be linked toa carrier that helps elicit an antibody response to the fragment and/orbe combined with an adjuvant that helps elicit such a response. Suchantibodies can be screened for differential binding to wild-type,monomeric versions of TTR or a fragment thereof (e.g., SEQ ID NO: 113)compared with mutants of specified residues. Screening against suchmutants more precisely defines the binding specificity to allowidentification of antibodies whose binding is inhibited by mutagenesisof particular residues and which are likely to share the functionalproperties of other exemplified antibodies. The mutations can besystematic replacement substitution with alanine (or serine if analanine is present already) one residue at a time, or more broadlyspaced intervals, throughout the target or throughout a section thereofin which an epitope is known to reside. If the same set of mutationssignificantly reduces the binding of two antibodies, the two antibodiesbind the same epitope.

Antibodies having the binding specificity of a selected murine antibody(e.g., 9D5 or 14G8) can also be produced using a variant of the phagedisplay method. See Winter, WO 92/20791. This method is particularlysuitable for producing human antibodies. In this method, either theheavy or light chain variable region of the selected murine antibody isused as a starting material. If, for example, a light chain variableregion is selected as the starting material, a phage library isconstructed in which members display the same light chain variableregion (i.e., the murine starting material) and a different heavy chainvariable region. The heavy chain variable regions can for example beobtained from a library of rearranged human heavy chain variableregions. A phage showing strong specific binding (e.g., at least 10⁸ andpreferably at least 10⁹ M⁻¹) for monomeric TTR or a fragment thereof(e.g., amino acid residues 89-97) is selected. The heavy chain variableregion from this phage then serves as a starting material forconstructing a further phage library. In this library, each phagedisplays the same heavy chain variable region (i.e., the regionidentified from the first display library) and a different light chainvariable region. The light chain variable regions can be obtained forexample from a library of rearranged human variable light chain regions.Again, phage showing strong specific binding for monomeric TTR or afragment thereof (e.g., amino acid residues 89-97) are selected. Theresulting antibodies usually have the same or similar epitopespecificity as the murine starting material. Kabat CDRs of the heavychain of 9D5 are designated SEQ ID NOS: 13-15, respectively, and KabatCDRs of the light chain of 9D5 are designated SEQ ID NOS: 24-26,respectively. A composite Chothia-Kabat CDR-H1 of 9D5 is designated SEQID NO: 117. Kabat CDRs of the heavy chain of 14G8 are designated SEQ IDNOS: 67-69, respectively, and Kabat CDRs of the light chain of 14G8 aredesignated SEQ ID NOS: 77-79, respectively. A composite Chothia-KabatCDR-H1 of 14G8 is designated SEQ ID NO: 118. A variant of CDR-L1 of 14G8is designated SEQ ID NO: 80.

Other antibodies can be obtained by mutagenesis of cDNA encoding theheavy and light chains of an exemplary antibody, such as 9D5 or 14G8.Monoclonal antibodies that are at least 70%, 80%, 90%, 95%, 96%, 97%,98%, or 99% identical to 9D5 or 14G8 in amino acid sequence of themature heavy and/or light chain variable regions and maintain itsfunctional properties, and/or which differ from the respective antibodyby a small number of functionally inconsequential amino acidsubstitutions (e.g., conservative substitutions), deletions, orinsertions are also included in the invention. Monoclonal antibodieshaving at least one or all six CDR(s) as defined by any conventionaldefinition, but preferably Kabat, that are 90%, 95%, 99% or 100%identical to corresponding CDRs of 9D5 or 14G8 are also included.

The invention also provides antibodies having some or all (e.g., 3, 4,5, and 6) CDRs entirely or substantially from 9D5 or 14G8. Suchantibodies can include a heavy chain variable region that has at leasttwo, and usually all three, CDRs entirely or substantially from theheavy chain variable region of 9D5 or 14G8 and/or a light chain variableregion having at least two, and usually all three, CDRs entirely orsubstantially from the light chain variable region of 9D5 or 14G8. Theantibodies can include both heavy and light chains. A CDR issubstantially from a corresponding 9D5 or 14G8 CDR when it contains nomore than 4, 3, 2, or 1 substitutions, insertions, or deletions, exceptthat CDR-H2 (when defined by Kabat) can have no more than 6, 5, 4, 3, 2,or 1 substitutions, insertions, or deletions. Such antibodies can haveat least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to 9D5 or14G8 in the amino acid sequence of the mature heavy and/or light chainvariable regions and maintain their functional properties, and/or differfrom 9D5 or 14G8 by a small number of functionally inconsequential aminoacid substitutions (e.g., conservative substitutions), deletions, orinsertions.

Some antibodies identified by such assays can bind to monomeric,misfolded, aggregated, or fibril forms of TTR but not to nativetetrameric forms of TTR, as described in the examples or otherwise.Likewise, some antibodies are immunoreactive on TTR-mediated amyloidosistissue but not on healthy tissue.

Some antibodies can inhibit or reduce aggregation of TTR, inhibit orreduce TTR fibril formation, reduce or clear TTR deposits or aggregatedTTR, or stabilize non-toxic conformations of TTR in an animal model orclinical trial. Some antibodies can treat, effect prophylaxis of, ordelay the onset of a TTR amyloidosis as shown in an animal model orclinical trial. Exemplary animal models for testing activity against aTTR amyloidosis include those described in Kohno et al., Am. J. Path.150(4):1497-1508 (1997); Teng et al., Laboratory Investigations81:385-396 (2001); Wakasugi et al., Proc. Japan Acad. 63B:344-347(1987); Shimada et al., Mol. Biol. Med. 6:333-343 (1989); Nagata et al.,J. Biochem. 117:169-175 (1995); Sousa et al., Am. J. Path. 161:1935-1948(2002); and Santos et al., Neurobiology of Aging 31:280-289 (2010).

B. Non-Human Antibodies

The production of other non-human antibodies, e.g., murine, guinea pig,primate, rabbit or rat, against monomeric TTR or a fragment thereof(e.g., amino acid residues 89-97) can be accomplished by, for example,immunizing the animal with TTR or a fragment thereof. See Harlow & Lane,Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated byreference for all purposes). Such an immunogen can be obtained from anatural source, by peptide synthesis, or by recombinant expression.Optionally, the immunogen can be administered fused or otherwisecomplexed with a carrier protein. Optionally, the immunogen can beadministered with an adjuvant. Several types of adjuvant can be used asdescribed below. Complete Freund's adjuvant followed by incompleteadjuvant is preferred for immunization of laboratory animals. Rabbits orguinea pigs are typically used for making polyclonal antibodies. Miceare typically used for making monoclonal antibodies. Antibodies arescreened for specific binding to monomeric TTR or an epitope within TTR(e.g., an epitope comprising one or more of amino acid residues 89-97).Such screening can be accomplished by determining binding of an antibodyto a collection of monomeric TTR variants, such as TTR variantscontaining amino acid residues 89-97 or mutations within these residues,and determining which TTR variants bind to the antibody. Binding can beassessed, for example, by Western blot, FACS or ELISA.

C. Humanized Antibodies

A humanized antibody is a genetically engineered antibody in which CDRsfrom a non-human “donor” antibody are grafted into human “acceptor”antibody sequences (see, e.g., Queen, U.S. Pat. Nos. 5,530,101 and5,585,089; Winter, U.S. Pat. No. 5,225,539; Carter, U.S. Pat. No.6,407,213; Adair, U.S. Pat. No. 5,859,205; and Foote, U.S. Pat. No.6,881,557). The acceptor antibody sequences can be, for example, amature human antibody sequence, a composite of such sequences, aconsensus sequence of human antibody sequences, or a germline regionsequence. Thus, a humanized antibody is an antibody having at leastthree, four, five or all CDRs entirely or substantially from a donorantibody and variable region framework sequences and constant regions,if present, entirely or substantially from human antibody sequences.Similarly a humanized heavy chain has at least one, two and usually allthree CDRs entirely or substantially from a donor antibody heavy chain,and a heavy chain variable region framework sequence and heavy chainconstant region, if present, substantially from human heavy chainvariable region framework and constant region sequences. Similarly ahumanized light chain has at least one, two and usually all three CDRsentirely or substantially from a donor antibody light chain, and a lightchain variable region framework sequence and light chain constantregion, if present, substantially from human light chain variable regionframework and constant region sequences. Other than nanobodies and dAbs,a humanized antibody comprises a humanized heavy chain and a humanizedlight chain. A CDR in a humanized antibody is substantially from acorresponding CDR in a non-human antibody when at least 85%, 90%, 95% or100% of corresponding residues (as defined by any conventionaldefinition but preferably defined by Kabat) are identical between therespective CDRs. The variable region framework sequences of an antibodychain or the constant region of an antibody chain are substantially froma human variable region framework sequence or human constant regionrespectively when at least 85%, 90%, 95% or 100% of correspondingresidues defined by any conventional definition but preferably definedby Kabat are identical.

Although humanized antibodies often incorporate all six CDRs (defined byany conventional definition but preferably as defined by Kabat) from amouse antibody, they can also be made with less than all CDRs (e.g., atleast 3, 4, or 5 CDRs) from a mouse antibody (e.g., Pascalis et al, J.Immunol. 169:3076, 2002; Vajdos et al., J. of Mol. Biol., 320: 415-428,2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al,J. Immunol., 164:1432-1441, 2000).

In some antibodies only part of the CDRs, namely the subset of CDRresidues required for binding, termed the SDRs, are needed to retainbinding in a humanized antibody. CDR residues not contacting antigen andnot in the SDRs can be identified based on previous studies (for exampleresidues H60-H65 in CDR H2 are often not required), from regions ofKabat CDRs lying outside Chothia hypervariable loops (Chothia, J. Mol.Biol. 196:901, 1987), by molecular modeling and/or empirically, or asdescribed in Gonzales et al., Mol. Immunol. 41: 863, 2004. In suchhumanized antibodies at positions in which one or more donor CDRresidues is absent or in which an entire donor CDR is omitted, the aminoacid occupying the position can be an amino acid occupying thecorresponding position (by Kabat numbering) in the acceptor antibodysequence. The number of such substitutions of acceptor for donor aminoacids in the CDRs to include reflects a balance of competingconsiderations. Such substitutions are potentially advantageous indecreasing the number of mouse amino acids in a humanized antibody andconsequently decreasing potential immunogenicity. However, substitutionscan also cause changes of affinity, and significant reductions inaffinity are preferably avoided. Positions for substitution within CDRsand amino acids to substitute can also be selected empirically.

The human acceptor antibody sequences can optionally be selected fromamong the many known human antibody sequences to provide a high degreeof sequence identity (e.g., 65-85% identity) between a human acceptorsequence variable region frameworks and corresponding variable regionframeworks of a donor antibody chain.

Examples of acceptor sequences for the heavy chain are the human matureheavy chain variable regions with NCBI accession codes BAC02114 andAAX82494.1 (SEQ ID NOS: 3 and 4) and heavy chain variable regions ofhuman Kabat subgroup 3. BAC02114 shares the same canonical form as mouse9D5 heavy chain. Other examples of acceptor sequences for the heavychain are the human mature heavy chain variable regions with NCBIaccession codes AAD30410.1 and AAX82494.1 (SEQ ID NOS: 63 and 4,respectively) and heavy chain variable regions of human Kabatsubgroup 1. AAD30410.1 and AAX82494.1 include two CDRs having the samecanonical form as mouse 14G8 heavy chain. Examples of acceptor sequencesfor the light chain are the human mature light chain variable regionwith NCBI accession code ABC66952 (SEQ ID NO: 18) and light chainvariable regions of human Kabat subgroup 3. ABC66952 includes two CDRshaving the same canonical form as mouse 9D5 light chain. Other examplesof acceptor sequences for the light chain are the human mature lightchain variable regions with NCBI accession codes ABA71374.1 andABC66952.1 (SEQ ID NOS: 72 and 73, respectively) and light chainvariable regions of human Kabat subgroup 2. ABA71374.1 and ABC66952.1have the same canonical form as mouse 14G8 light chain.

If more than one human acceptor antibody sequence is selected, acomposite or hybrid of those acceptors can be used, and the amino acidsused at different positions in the humanized light chain and heavy chainvariable regions can be taken from any of the human acceptor antibodysequences used. For example, the human mature heavy chain variableregions with NCBI accession codes BAC02114 and AAX82494.1 (SEQ ID NOS: 3and 4) were used as acceptor sequences for humanization of the 9D5mature heavy chain variable region. Examples of positions in which thesetwo acceptors differ include positions H19 (R or K), H40 (A or T), H44(G or R), H49 (S or A), H77 (S or T), H82a (N or S), H83 (R or K), H84(A or S), and H89 (V or M). Humanized versions of the 9D5 heavy chainvariable region can include either amino acid at any of these positions.Similarly, the human mature heavy chain variable regions with NCBIaccession codes AAD30410.1 and AAX82494.1 (SEQ ID NOS: 63 and 4,respectively) were used as acceptor sequences for humanization of the14G8 mature heavy chain variable region. Examples of positions in whichthese two acceptors differ include positions H82a (N or S), H83 (R orK), H84 (A or S), and H89 (V or M). Humanized versions of the 14G8 heavychain variable region can include either amino acid at any of thesepositions. Similarly, the human mature light chain variable regions withNCBI accession codes ABA71374.1 and ABC66952.1 (SEQ ID NOS: 72 and 73,respectively) were used as acceptor sequences for humanization of the14G8 mature light chain variable region. An example of a position inwhich these two acceptors differ is position L18 (S or P). Humanizedversions of the 14G8 light chain variable region can include eitheramino acid at this position.

Certain amino acids from the human variable region framework residuescan be selected for substitution based on their possible influence onCDR conformation and/or binding to antigen. Investigation of suchpossible influences is by modeling, examination of the characteristicsof the amino acids at particular locations, or empirical observation ofthe effects of substitution or mutagenesis of particular amino acids.

For example, when an amino acid differs between a murine variable regionframework residue and a selected human variable region frameworkresidue, the human framework amino acid can be substituted by theequivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

-   -   (1) noncovalently binds antigen directly;    -   (2) is adjacent to a CDR region or within a CDR as defined by        Chothia but not Kabat;    -   (3) otherwise interacts with a CDR region (e.g., is within about        6 Å of a CDR region), (e.g., identified by modeling the light or        heavy chain on the solved structure of a homologous known        immunoglobulin chain); or    -   (4) is a residue participating in the VL-VH interface.

Framework residues from classes (1) through (3) as defined by Queen,U.S. Pat. No. 5,530,101, are sometimes alternately referred to ascanonical and vernier residues. Framework residues that help define theconformation of a CDR loop are sometimes referred to as canonicalresidues (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Thornton &Martin, J. Mol. Biol. 263:800-815 (1996)). Framework residues thatsupport antigen-binding loop conformations and play a role infine-tuning the fit of an antibody to antigen are sometimes referred toas vernier residues (Foote & Winter, J. Mol. Biol 224:487-499 (1992)).

Other framework residues that are candidates for substitution areresidues creating a potential glycosylation site. Still other candidatesfor substitution are acceptor human framework amino acids that areunusual for a human immunoglobulin at that position. These amino acidscan be substituted with amino acids from the equivalent position of themouse donor antibody or from the equivalent positions of more typicalhuman immunoglobulins.

Exemplary humanized antibodies are humanized forms of the mouse 9D5 or14G8 antibodies, designated Hu9D5 or Hu14G8, respectively. The mouse 9D5antibody comprises mature heavy and light chain variable regions havingamino acid sequences comprising SEQ ID NO: 1 and SEQ ID NO: 16,respectively. The invention provides eight exemplified humanized matureheavy chain variable regions: Hu9D5VHv1, Hu9D5VHv2, Hu9D5VHv2b,Hu9D5VHv3, Hu9D5VHv3b, Hu9D5VHv4, Hu9D5VHv4b, and Hu9D5VHv5 (SEQ ID NOS:5-12, respectively). The invention further provides five exemplifiedhuman mature light chain variable regions: Hu9D5VLv1, Hu9D5VLv2,Hu9D5VLv3, Hu9D5VLv4, and Hu9D5VLv5 (SEQ ID NOS: 19-23, respectively).FIG. 1 shows alignments of 9D5, mouse model antibodies, human acceptorantibodies, and various humanized antibodies.

The mouse 14G8 antibody comprises mature heavy and light chain variableregions having amino acid sequences comprising SEQ ID NO: 61 and SEQ IDNO: 70, respectively. The invention provides three exemplified humanizedmature heavy chain variable regions: Hu14G8VHv1, Hu14G8VHv2, andHu14G8VHv3 (SEQ ID NOS: 64-66, respectively). The invention furtherprovides three exemplified human mature light chain variable regions:Hu14G8VLv1, Hu14G8VLv2, and Hu14G8VLv3 (SEQ ID NOS: 74-76,respectively). FIG. 2 shows alignments of 14G8, mouse model antibodies,human acceptor antibodies, and various humanized antibodies.

For reasons such as possible influence on CDR conformation and/orbinding to antigen, mediating interaction between heavy and lightchains, interaction with the constant region, being a site for desiredor undesired post-translational modification, being an unusual residuefor its position in a human variable region sequence and thereforepotentially immunogenic, getting aggregation potential, and otherreasons, the following 15 variable region framework positions wereconsidered as candidates for substitutions in the eight exemplifiedHu9D5 mature heavy chain variable regions and the five exemplified Hu9D5mature light chain variable regions, as further specified in theexamples: H42 (G42E), H47 (W47L), H69 (I69F), H82 (M82S), H82b(S82(b)L), H108 (T108L), L8 (P8A), L9 (L9P), L18 (P18S), L19 (A19V), L36(Y36F), L39 (K39R), L60 (D60S), L70 (D70A), and L74 (K74R). Likewise,the following 11 variable region framework positions were considered ascandidates for substitutions in the three exemplified Hu14G8 matureheavy chain variable regions and the three exemplified Hu14G8 maturelight chain variable regions, as further specified in the examples: H1(Q1E), H3 (Q3K), H47 (W47L), H105 (Q105T), L8 (P8A), L9 (L9P), L19(A19V), L26 (N26S), L36 (Y36F), L60 (D60S), and L70 (D70A).

Here, as elsewhere, the first-mentioned residue is the residue of ahumanized antibody formed by grafting Kabat CDRs or a compositeChothia-Kabat CDR in the case of CDR-H1 into a human acceptor framework(e.g., a composite or hybrid human acceptor framework), and thesecond-mentioned residue is a residue being considered for replacingsuch residue. Thus, within variable region frameworks, the firstmentioned residue is human, and within CDRs, the first mentioned residueis mouse.

Exemplified Hu9D5 antibodies include any permutations or combinations ofthe exemplified mature heavy and light chain variable regions (e.g.,VHv1/VLv1 or H1L1, VHv1/VLv2 or H1L2, VHv1/VLv3 or H1L3, VHv1/VLv4 orH1L4, VHv1/VLv5 or H1L5, VHv2/VLv1 or H2L1, VHv2/VLv2 or H2L2, VHv2/VLv3or H2L3, VHv2/VLv4 or H2L4, VHv2/VLv5 or H2L5, VHv2b/VLv1 or H2bL1,VHv2b/VLv2 or H2bL2, VHv2b/VLv3 or H2bL3, VHv2b/VLv4 or H2bL4,VHv2b/VLv5 or H2bL5, VHv3/VLv1 or H3L1, VHv3/VLv2 or H3L2, VHv3/VLv3 orH3L3, VHv3/VLv4 or H3L4, VHv3/VLv5 or H3L5, VHv3b/VLv1 or H3bL1,VHv3b/VLv2 or H3bL2, VHv3b/VLv3 or H3bL3, VHv3b/VLv4 or H3bL4,VHv3b/VLv5 or H3bL5, VHv4/VLv1 or H4L1, VHv4/VLv2 or H4L2, VHv4/VLv3 orH4L3, VHv4/VLv4 or H4L4, VHv4/VLv5 or H4L5, VHv4b/VLv1 or H4bL1,VHv4b/VLv2 or H4bL2, VHv4b/VLv3 or H4bL3, VHv4b/VLv4 or H4bL4,VHv4b/VLv5 or H4bL5, VHv5/VLv1 or H5L1, VHv5/VLv2 or H5L2, VHv5/VLv3 orH5L3, VHv5/VLv4 or H5L4, and VHv5/VLv5 or H5L5).

The invention provides variants of humanized 9D5 antibodies in which thehumanized mature heavy chain variable region shows at least 90%, 95%,96%, 97%, 98%, or 99% identity to a humanized Hu9D5VHv4b (SEQ ID NO: 11)and the humanized mature light chain variable region shows at least 90%,95%, 96%, 97%, 98%, or 99% identity to a Hu9D5VLv1 (SEQ ID NO: 19). Insome such antibodies, at least 1, 2, or all 3 of the backmutations orother mutations in Hu9D5 H4bL1 are retained. The invention also providesvariants of the other exemplified humanized 9D5 antibodies. Suchvariants have mature light and heavy chain variable regions showing atleast 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the maturelight and heavy chain variable regions of the exemplified humanized 9D5H1L1, H1L2, H1L3, H1L4, H1L5, H2L1, H2L2, H2L3, H2L4, H2L5, H2bL1,H2bL2, H2bL3, H2bL4, H2bL5, H3L1, H3L2, H3L3, H3L4, H3L5, H3bL1, Hb3L2,H3bL3, Hb3L4, H3bL5, H4L1, H4L2, H4L3, H4L4, H4L5, H4bL1, H4bL2, H4bL3,H4bL4, H4bL5, H5L1, H5L2, H5L3, H5L4, or H5L5 antibodies.

In some antibodies, at least one of positions H42, H47, H69, H82, H82b,and H108 in the Vh region is occupied by E, L, F, S, L, and L,respectively. In some antibodies, positions H47, H69, and H82 in the Vhregion are occupied by L, F, and S, respectively, as in Hu9D5VHv1. Insome antibodies, positions H47, H69, H82, and H82b in the Vh region areoccupied by L, F, S, and L, respectively, as in Hu9D5VHv2. In someantibodies, positions H42, H47, and H108 in the Vh region are occupiedby E, L, and L, respectively, as in Hu9D5VHv2b. In some antibodies,positions H69, H82, and H82b in the Vh region are occupied by F, S, andL, respectively, as in Hu9D5VHv3. In some antibodies, positions H47 andH108 in the Vh region are each occupied by L, as in Hu9D5VHv3b andHu9D5Vhv4b. In some antibodies, positions H82 and H82b in the Vh regionare occupied by S and L, respectively, as in Hu9D5VHv4. In someantibodies, positions H42, H47, and H82b in the Vh region are occupiedby E, L, and L, respectively, as in Hu9D5VHv5. In some antibodies, atleast one of positions L8, L9, L18, L19, L36, L39, L60, L70, and L74 inthe Vk region is occupied by A, P, S, V, F, R, S, A, and R,respectively. In some antibodies, position L36 in the Vk region isoccupied by F, as in Hu9D5VLv1. In some antibodies, position L60 in theVk region is occupied by S, as in Hu9D5VLv3. In some antibodies,positions L8, L9, L19, L36, L39, L60, L70, and L74 in the Vk region areoccupied by A, P, V, F, R, S, A, and R, respectively, as in Hu9D5VLv4.In some antibodies, positions L8, L9, L18, L19, L36, L39, L60, L70, andL74 in the Vk region are occupied by A, P, S, V, F, R, S, A, and R,respectively, as in Hu9D5VLv5. The CDR regions of such humanizedantibodies can be identical or substantially identical to the CDRregions of the 9D5 mouse donor antibody or any of the above exemplifiedhumanized 9D5 antibodies. The CDR regions can be defined by anyconventional definition (e.g., Chothia, or composite of Chothia andKabat) but are preferably as defined by Kabat.

Variable regions framework positions are in accordance with Kabatnumbering unless otherwise stated. Other such variants typically differfrom the sequences of the exemplified Hu9D5 heavy and light chains by asmall number (e.g., typically no more than 1, 2, 3, 5, 10, or 15) ofreplacements, deletions or insertions. Such differences are usually inthe framework but can also occur in the CDRs.

Exemplified Hu14G8 antibodies include any permutations or combinationsof the exemplified mature heavy and light chain variable regions (e.g.,VHv1/VLv1 or H1L1, VHv1/VLv2 or H1L2, VHv1/VLv3 or H1L3, VHv2/VLv1 orH2L1, VHv2/VLv2 or H2L2, VHv2/VLv3 or H2L3, VHv3/VLv1 or H3L1, VHv3/VLv2or H3L2, and VHv3/VLv3 or H3L3).

The invention provides variants of humanized 14G8 antibodies in whichthe humanized mature heavy chain variable region shows at least 90%,95%, 96%, 97%, 98%, or 99% identity to Hu14G8VHv2 (Hu14G8 H2) (SEQ IDNO: 65) and the humanized mature light chain variable region shows atleast 90%, 95%, 96%, 97%, 98%, or 99% identity to Hu14G8VLv3 (Hu14G8 L3)(SEQ ID NO: 76). In some such antibodies, at least 1, 2, 3, 4, or all 5of the backmutations or other mutations in Hu14G8 H2L3 are retained. Theinvention also provides variants of the other exemplified humanized 14G8antibodies. Such variants have mature light and heavy chain variableregions showing at least 90%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to the mature light and heavy chain variable regions of theexemplified humanized 14G8 H1L1, H1L2, H1L3, H2L1, H2L2, H2L3, H3L1,H3L2, or H3L3 antibodies.

In some antibodies, at least one of positions H1 and H47 in the Vhregion is occupied by E and L, respectively. In some antibodies,positions H1 and H47 in the Vh region are occupied by E and L,respectively, as in Hui4G8VHv2 and Hui4G8VHv3. In some antibodies, atleast one of positions H3 and H105 in the Vh region is occupied by K andT, respectively. In some antibodies, positions H3 and H105 in the Vhregion are occupied by K and T, respectively, as in Hu14G8VHv1. In someantibodies, position L36 in the Vk region is occupied by F, as inHu14G8VLv2. In some antibodies, at least one of positions L8, L9, L19,L26, L60, and L70 in the Vk region is occupied by A, P, V, S, S, and A,respectively. In some antibodies, positions L8, L9, L19, and L70 in theVk region are occupied by A, P, V, and A, respectively, as inHu14G8VLv1. In some antibodies, positions L26 and L60 in the Vk regionare each occupied by S, as in Hu14G8VLv3. The CDR regions of suchhumanized antibodies can be identical or substantially identical to theCDR regions of the 14G8 mouse donor antibody. The CDR regions can bedefined by any conventional definition (e.g., Chothia, or composite ofChothia and Kabat) but are preferably as defined by Kabat.

Variable regions framework positions are in accordance with Kabatnumbering unless otherwise stated. Other such variants typically differfrom the sequences of the exemplified Hu14G8 heavy and light chains by asmall number (e.g., typically no more than 1, 2, 3, 5, 10, or 15) ofreplacements, deletions or insertions. Such differences are usually inthe framework but can also occur in the CDRs

A possibility for additional variation in humanized 9D5 or 14G8 variantsis additional backmutations in the variable region frameworks. Many ofthe framework residues not in contact with the CDRs in the humanized mAbcan accommodate substitutions of amino acids from the correspondingpositions of the donor mouse mAb or other mouse or human antibodies, andeven many potential CDR-contact residues are also amenable tosubstitution. Even amino acids within the CDRs may be altered, forexample, with residues found at the corresponding position of the humanacceptor sequence used to supply variable region frameworks. Inaddition, alternate human acceptor sequences can be used, for example,for the heavy and/or light chain. If different acceptor sequences areused, one or more of the backmutations recommended above may not beperformed because the corresponding donor and acceptor residues arealready the same without backmutations.

Preferably, replacements or backmutations in humanized 9D5 or 14G8variants (whether or not conservative) have no substantial effect on thebinding affinity or potency of the humanized mAb, that is, its abilityto bind to monomeric TTR (e.g., the potency in some or all of the assaysdescribed in the present examples of the variant humanized 9D5 or 14G8antibody is essentially the same, i.e., within experimental error, asthat of murine 9D5 or 14G8 antibody).

D. Chimeric and Veneered Antibodies

The invention further provides chimeric and veneered forms of non-humanantibodies, particularly the 9D5 or 14G8 antibodies of the examples.

A chimeric antibody is an antibody in which the mature variable regionsof light and heavy chains of a non-human antibody (e.g., a mouse) arecombined with human light and heavy chain constant regions. Suchantibodies substantially or entirely retain the binding specificity ofthe mouse antibody, and are about two-thirds human sequence.

A veneered antibody is a type of humanized antibody that retains someand usually all of the CDRs and some of the non-human variable regionframework residues of a non-human antibody but replaces other variableregion framework residues that may contribute to B- or T-cell epitopes,for example exposed residues (Padlan, Mol Immunol. 28:489, 1991) withresidues from the corresponding positions of a human antibody sequence.The result is an antibody in which the CDRs are entirely orsubstantially from a non-human antibody and the variable regionframeworks of the non-human antibody are made more human-like by thesubstitutions. Veneered forms of the 9D5 or 14G8 antibody are includedin the invention.

E. Human Antibodies

Human antibodies against monomeric TTR or a fragment thereof (e.g.,amino acid residues 89-97 (SEQ ID NO: 113) of TTR) are provided by avariety of techniques described below. Some human antibodies areselected by competitive binding experiments, by the phage display methodof Winter, above, or otherwise, to have the same epitope specificity asa particular mouse antibody, such as one of the mouse monoclonalantibodies described in the examples. Human antibodies can also bescreened for particular epitope specificity by using only a fragment ofTTR, such as a TTR variant containing only amino acid residues 89-97 ofTTR, as the target antigen, and/or by screening antibodies against acollection of TTR variants, such as TTR variants containing variousmutations within amino acid residues 89-97 of TTR.

Methods for producing human antibodies include the trioma method ofOestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Pat. No.4,634,664; and Engleman et al., U.S. Pat. No. 4,634,666, use oftransgenic mice including human immunoglobulin genes (see, e.g., Lonberget al, WO93/12227 (1993); U.S. Pat. Nos. 5,877,397; 5,874,299;5,814,318; 5,789,650; 5,770,429; 5,661,016; 5,633,425; 5,625,126;5,569,825; 5,545,806; Neuberger, Nat. Biotechnol 14:826 (1996); andKucherlapati, WO 91/10741 (1991)) and phage display methods (see, e.g.,Dower et al., WO 91/17271; McCafferty et al., WO 92/01047; U.S. Pat.Nos. 5,877,218; 5,871,907; 5,858,657; 5,837,242; 5,733,743; and5,565,332).

F. Selection of Constant Region

The heavy and light chain variable regions of chimeric, veneered orhumanized antibodies can be linked to at least a portion of a humanconstant region. The choice of constant region depends, in part, whetherantibody-dependent cell-mediated cytotoxicity, antibody dependentcellular phagocytosis and/or complement dependent cytotoxicity aredesired. For example, human isotopes IgG1 and IgG3 havecomplement-dependent cytotoxicity and human isotypes IgG2 and IgG4 donot. Human IgG1 and IgG3 also induce stronger cell mediated effectorfunctions than human IgG2 and IgG4. Light chain constant regions can belambda or kappa.

One or several amino acids at the amino or carboxy terminus of the lightand/or heavy chain, such as the C-terminal lysine of the heavy chain,may be missing or derivatized in a proportion or all of the molecules.Substitutions can be made in the constant regions to reduce or increaseeffector function such as complement-mediated cytotoxicity or ADCC (see,e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No.5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006),or to prolong half-life in humans (see, e.g., Hinton et al., J. Biol.Chem. 279:6213, 2004). Exemplary substitutions include a G1n at position250 and/or a Leu at position 428 (EU numbering is used in this paragraphfor the constant region) for increasing the half-life of an antibody.Substitution at any or all of positions 234, 235, 236 and/or 237 reduceaffinity for Fcγ receptors, particularly FcγRI receptor (see, e.g., U.S.Pat. No. 6,624,821). An alanine substitution at positions 234, 235, and237 of human IgG1 can be used for reducing effector functions. Someantibodies have alanine substitution at positions 234, 235 and 237 ofhuman IgG1 for reducing effector functions. Optionally, positions 234,236 and/or 237 in human IgG2 are substituted with alanine and position235 with glutamine (see, e.g., U.S. Pat. No. 5,624,821). In someantibodies, a mutation at one or more of positions 241, 264, 265, 270,296, 297, 322, 329, and 331 by EU numbering of human IgG1 is used. Insome antibodies, a mutation at one or more of positions 318, 320, and322 by EU numbering of human IgG1 is used. In some antibodies, positions234 and/or 235 are substituted with alanine and/or position 329 issubstituted with glycine. In some antibodies, positions 234 and 235 aresubstituted with alanine, such as in SEQ ID NO: 102. In some antibodies,the isotype is human IgG2 or IgG4.

An exemplary human light chain kappa constant region has the amino acidsequence of SEQ ID NO: 104. The N-terminal arginine of SEQ ID NO: 104can be omitted, in which case light chain kappa constant region has theamino acid sequence of SEQ ID NO: 105. An exemplary human IgG1 heavychain constant region has the amino acid sequence of SEQ ID NO: 101(with or without the C-terminal lysine). Antibodies can be expressed astetramers containing two light and two heavy chains, as separate heavychains, light chains, as Fab, Fab′, F(ab′)2, and Fv, or as single chainantibodies in which heavy and light chain mature variable domains arelinked through a spacer.

Human constant regions show allotypic variation and isoallotypicvariation between different individuals, that is, the constant regionscan differ in different individuals at one or more polymorphicpositions. Isoallotypes differ from allotypes in that sera recognizingan isoallotype bind to a non-polymorphic region of a one or more otherisotypes. Thus, for example, another heavy chain constant region is ofIgG1 G1m3 allotype and has the amino acid sequence of SEQ ID NO: 103.Another heavy chain constant region of the IgG1 G1m3 allotype has theamino acid sequence of SEQ ID NO: 102 (with or without the C-terminallysine). Reference to a human constant region includes a constant regionwith any natural allotype or any permutation of residues occupyingpositions in natural allotypes.

G. Expression of Recombinant Antibodies

A number of methods are known for producing chimeric and humanizedantibodies using an antibody-expressing cell line (e.g., hybridoma). Forexample, the immunoglobulin variable regions of antibodies can be clonedand sequenced using well known methods. In one method, the heavy chainvariable VH region is cloned by RT-PCR using mRNA prepared fromhybridoma cells. Consensus primers are employed to the VH region leaderpeptide encompassing the translation initiation codon as the 5′ primerand a g2b constant regions specific 3′ primer. Exemplary primers aredescribed in U.S. patent publication US 2005/0009150 by Schenk et al.(hereinafter “Schenk”). The sequences from multiple, independentlyderived clones can be compared to ensure no changes are introducedduring amplification. The sequence of the VH region can also bedetermined or confirmed by sequencing a VH fragment obtained by 5′ RACERT-PCR methodology and the 3′ g2b specific primer.

The light chain variable VL region can be cloned in an analogous manner.In one approach, a consensus primer set is designed for amplification ofVL regions using a 5′ primer designed to hybridize to the VL regionencompassing the translation initiation codon and a 3′ primer specificfor the Ck region downstream of the V-J joining region. In a secondapproach, 5′RACE RT-PCR methodology is employed to clone a VL encodingcDNA. Exemplary primers are described in Schenk, supra. The clonedsequences are then combined with sequences encoding human (or othernon-human species) constant regions. Exemplary sequences encoding humanconstant regions include SEQ ID NO: 106, which encodes a human IgG1constant region (SEQ ID NO: 103), and SEQ ID NOS: 107 and 108, whichencode human kappa light chain constant regions (SEQ ID NOS: 104 and105, respectively).

In one approach, the heavy and light chain variable regions arere-engineered to encode splice donor sequences downstream of therespective VDJ or VJ junctions and are cloned into a mammalianexpression vector, such as pCMV-hyl for the heavy chain and pCMV-Mcl forthe light chain. These vectors encode human γ1 and Ck constant regionsas exonic fragments downstream of the inserted variable region cassette.Following sequence verification, the heavy chain and light chainexpression vectors can be co-transfected into CHO cells to producechimeric antibodies. Conditioned media is collected 48 hourspost-transfection and assayed by western blot analysis for antibodyproduction or ELISA for antigen binding. The chimeric antibodies arehumanized as described above.

Chimeric, veneered, humanized, and human antibodies are typicallyproduced by recombinant expression. Recombinant polynucleotideconstructs typically include an expression control sequence operablylinked to the coding sequences of antibody chains, including naturallyassociated or heterologous expression control elements, such as apromoter. The expression control sequences can be promoter systems invectors capable of transforming or transfecting eukaryotic orprokaryotic host cells. Once the vector has been incorporated into theappropriate host, the host is maintained under conditions suitable forhigh level expression of the nucleotide sequences and the collection andpurification of the crossreacting antibodies.

These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors contain selection markers, e.g., ampicillinresistance or hygromycin resistance, to permit detection of those cellstransformed with the desired DNA sequences.

E. coli is one prokaryotic host useful for expressing antibodies,particularly antibody fragments. Microbes, such as yeast, are alsouseful for expression. Saccharomyces is a yeast host with suitablevectors having expression control sequences, an origin of replication,termination sequences, and the like as desired. Typical promotersinclude 3-phosphoglycerate kinase and other glycolytic enzymes.Inducible yeast promoters include, among others, promoters from alcoholdehydrogenase, isocytochrome C, and enzymes responsible for maltose andgalactose utilization.

Mammalian cells can be used for expressing nucleotide segments encodingimmunoglobulins or fragments thereof. See Winnacker, From Genes toClones, (VCH Publishers, NY, 1987). A number of suitable host cell linescapable of secreting intact heterologous proteins have been developed,and include CHO cell lines, various COS cell lines, HeLa cells, HEK293cells, L cells, and non-antibody-producing myelomas including Sp2/0 andNS0. The cells can be nonhuman. Expression vectors for these cells caninclude expression control sequences, such as an origin of replication,a promoter, an enhancer (Queen et al, Immunol. Rev. 89:49 (1986)), andnecessary processing information sites, such as ribosome binding sites,RNA splice sites, polyadenylation sites, and transcriptional terminatorsequences. Expression control sequences can include promoters derivedfrom endogenous genes, cytomegalovirus, SV40, adenovirus, bovinepapillomavirus, and the like. See Co et al, J. Immunol. 148:1149 (1992).

Alternatively, antibody coding sequences can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal (see, e.g.,U.S. Pat. Nos. 5,741,957; 5,304,489; and 5,849,992). Suitable transgenesinclude coding sequences for light and/or heavy chains operably linkedwith a promoter and enhancer from a mammary gland specific gene, such ascasein or beta lactoglobulin.

The vectors containing the DNA segments of interest can be transferredinto the host cell by methods depending on the type of cellular host.For example, calcium chloride transfection is commonly utilized forprokaryotic cells, whereas calcium phosphate treatment, electroporation,lipofection, biolistics, or viral-based transfection can be used forother cellular hosts. Other methods used to transform mammalian cellsinclude the use of polybrene, protoplast fusion, liposomes,electroporation, and microinjection. For production of transgenicanimals, transgenes can be microinjected into fertilized oocytes or canbe incorporated into the genome of embryonic stem cells, and the nucleiof such cells transferred into enucleated oocytes.

Having introduced vector(s) encoding antibody heavy and light chainsinto cell culture, cell pools can be screened for growth productivityand product quality in serum-free media. Top-producing cell pools canthen be subjected of FACS-based single-cell cloning to generatemonoclonal lines. Specific productivities above 50 pg or 100 pg per cellper day, which correspond to product titers of greater than 7.5 g/Lculture, can be used. Antibodies produced by single cell clones can alsobe tested for turbidity, filtration properties, PAGE, IEF, UV scan,HP-SEC, carbohydrate-oligosaccharide mapping, mass spectrometry, andbinding assay, such as ELISA or Biacore. A selected clone can then bebanked in multiple vials and stored frozen for subsequent use.

Once expressed, antibodies can be purified according to standardprocedures of the art, including protein A capture, HPLC purification,column chromatography, gel electrophoresis and the like (see generally,Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

Methodology for commercial production of antibodies can be employed,including codon optimization, selection of promoters, selection oftranscription elements, selection of terminators, serum-free single cellcloning, cell banking, use of selection markers for amplification ofcopy number, CHO terminator, or improvement of protein titers (see,e.g., U.S. Pat. Nos. 5,786,464; 6,114,148; 6,063,598; 7,569,339;WO2004/050884; WO2008/012142; WO2008/012142; WO2005/019442;WO2008/107388; WO2009/027471; and U.S. Pat. No. 5,888,809).

H. Antibody Screening Assays

Antibodies can be subject to several screens including binding assays,functional screens, screens in animal models of diseases associated withTTR deposits, and clinical trials. Binding assays test for specificbinding and, optionally, affinity and epitope specificity to monomericTTR or a fragment thereof. For example, binding assays can screen forantibodies that bind to amino acid residues 89-97 (SEQ ID NO: 113) ofTTR, which is an epitope that is buried in the native TTR tetramer andexposed in monomeric, misfolded, aggregated, or fibril forms of TTR.Antibodies can also be screened for the ability to bind pre-fibrillar,non-native conformations of TTR and TTR amyloid fibrils but not nativeTTR conformations. For example, antibodies can be screened for theability to bind to monomeric forms of TTR created by dissociation ordisaggregation of native tetrameric TTR, and can be counter-screenedagainst native tetrameric TTR, as described in the examples orotherwise. Likewise, antibodies can also be screened for theirimmunoreactivity on TTR-mediated amyloidosis tissue but not on healthytissue. Such screens are sometimes performed in competition with anexemplary antibody, such as an antibody having the variable regions of9D5 or 14G8 (IgG1 kappa isotype). Optionally, either the antibody or TTRtarget is immobilized in such assay.

Functional assays can be performed in cellular models including cellsnaturally expressing TTR or transfected with DNA encoding TTR or afragment thereof. Suitable cells include cells derived from cardiactissue or other tissues affected by TTR amyloidogenesis. Cells can bescreened for reduced levels of monomeric, misfolded, aggregated, orfibril forms of TTR (e.g., by Western blotting or immunoprecipitation ofcell extracts or supernatants) or reduced toxicity attributable tomonomeric, misfolded, aggregated, or fibril forms of TTR. For example,antibodies can tested for the ability to inhibit or reduce aggregationof TTR, inhibit or reduce TTR fibril formation, reduce TTR deposits,clear aggregated TTR, or stabilize non-toxic conformations of TTR.

Other functional assays can be performed in solution, such as testingwhether an antibody is capable of disrupting or reducing TTR fibrilformation when monomeric TTR or misfolded TTR intermediates in solutionare contacted with the antibody. The extent of fibril formation can beprobed by turbidity measurements, for example, at 400 nm on a UV-visiblespectrometer equipped with a temperature control unit. Thioflavin-T canalso be used to assess the extent of amyloid fibril formation. Forexample, a five-fold molar excess of Thioflavin-T can be added to TTRsamples and left at room temperature for 30 minutes before measurementsare taken. Thioflavin-T fluorescence can be monitored using aspectrofluorimeter. See US 2014/0056904.

Animal model screens test the ability of the antibody to therapeuticallyor prophylactically treat signs or symptoms in an animal modelsimulating a human disease associated with accumulation of TTR or TTRdeposits. Such diseases include types of TTR amyloidosis, such as senilesystemic amyloidosis (SSA), senile cardiac amyloidosis (SCA), familialamyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), andcentral nervous system selective amyloidosis (CNSA). Suitable signs orsymptoms that can be monitored include the presence and extent ofamyloid deposits in various tissues, such as the gastrointestinal tractor heart. The extent of reduction of amyloid deposits can be determinedby comparison with an appropriate control, such the level of TTR amyloiddeposits in control animals that have received a control antibody (e.g.,an isotype matched control antibody), a placebo, or no treatment at all.An exemplary animal model for testing activity against a TTR amyloidosisis a mouse model carrying a null mutation at the endogenous mouse Ttrlocus and the human mutant TTR gene comprising a V30M mutation that isassociated with familial amyloidotic polyneuropathy. See, e.g., Kohno etal., Am. J. Path. 150(4):1497-1508 (1997); Cardoso and Saraiva, FASEB J20(2):234-239 (2006). Similar models also exist, including other modelsfor familial versions of TTR amyloidosis and models for sporadicversions of TTR amyloidosis. See, e.g., Teng et al., Lab. Invest. 81(3):385-396 (2001); Ito and Maeda, Mouse Models of TransthyretinAmyloidosis, in Recent Advances in Transthyretin Evolution, Structure,and Biological Functions, pp. 261-280 (2009) (Springer BerlinHeidelberg). Transgenic animals can include a human TTR transgene, suchas a TTR transgene with a mutation associated with TTR amyloidosis or awild-type TTR transgene. To facilitate testing in animal models,chimeric antibodies having a constant region appropriate for the animalmodel can be used (e.g., mouse-rat chimeras could be used for testingantibodies in rats). It can be concluded that a humanized version of anantibody will be effective if the corresponding mouse antibody orchimeric antibody is effective in an appropriate animal model and thehumanized antibody has similar binding affinity (e.g., withinexperimental error, such as by a factor of 1.5, 2, or 3).

Clinical trials test for safety and efficacy in a human having a diseaseassociated with TTR amyloidosis.

I. Nucleic Acids

The invention further provides nucleic acids encoding any of the heavyand light chains described above (e.g., SEQ ID NOS: 40, 42, 44-56, 87,89, 91-96, and 106-108). Optionally, such nucleic acids further encode asignal peptide and can be expressed with the signal peptide linked tothe constant region (e.g., signal peptides having amino acid sequencesof SEQ ID NOS: 41 and 88 (heavy chain) and 43 and 90 (light chain) thatcan be encoded by SEQ ID NOS: 40 and 87, respectively (heavy chain) and42 and 89, respectively (light chain)). Coding sequences of nucleicacids can be operably linked with regulatory sequences to ensureexpression of the coding sequences, such as a promoter, enhancer,ribosome binding site, transcription termination signal, and the like.The nucleic acids encoding heavy and light chains can occur in isolatedform or can be cloned into one or more vectors. The nucleic acids can besynthesized by, for example, solid state synthesis or PCR of overlappingoligonucleotides. Nucleic acids encoding heavy and light chains can bejoined as one contiguous nucleic acid, e.g., within an expressionvector, or can be separate, e.g., each cloned into its own expressionvector.

J. Conjugated Antibodies

Conjugated antibodies that specifically bind to antigens exposed inpathogenic forms of TTR but not in native tetrameric forms of TTR, suchas amino acid residues 89-97 (SEQ ID NO: 113) of TTR, are useful indetecting the presence of monomeric, misfolded, aggregated, or fibrilforms of TTR; monitoring and evaluating the efficacy of therapeuticagents being used to treat patients diagnosed with a TTR amyloidosis;inhibiting or reducing aggregation of TTR; inhibiting or reducing TTRfibril formation; reducing or clearing TTR deposits; stabilizingnon-toxic conformations of TTR; or treating or effecting prophylaxis ofa TTR amyloidosis in a patient. For example, such antibodies can beconjugated with other therapeutic moieties, other proteins, otherantibodies, and/or detectable labels. See WO 03/057838; U.S. Pat. No.8,455,622.

Conjugated therapeutic moieties can be any agent that can be used totreat, combat, ameliorate, prevent, or improve an unwanted condition ordisease in a patient, such as a TTR amyloidosis. Therapeutic moietiescan include, for example, immunomodulators or any biologically activeagents that facilitate or enhance the activity of the antibody. Animmunomodulator can be any agent that stimulates or inhibits thedevelopment or maintenance of an immunologic response. If suchtherapeutic moieties are coupled to an antibody specific for monomeric,misfolded, aggregated, or fibril forms of TTR, such as the antibodiesdescribed herein, the coupled therapeutic moieties will have a specificaffinity for non-native, pathogenic forms of TTR over native tetramericforms of TTR. Consequently, administration of the conjugated antibodiesdirectly targets tissues comprising pathogenic forms of TTR with minimaldamage to surrounding normal, healthy tissue. This can be particularlyuseful for therapeutic moieties that are too toxic to be administered ontheir own. In addition, smaller quantities of the therapeutic moietiescan be used.

Examples of suitable therapeutic moieties include drugs that reducelevels of TTR, stabilize the native tetrameric structure of TTR, inhibitaggregation of TTR, disrupt TTR fibril or amyloid formation, orcounteract cellular toxicity. See, e.g., Almeida and Saraiva, FEBSLetters 586:2891-2896 (2012); Saraiva, FEBS Letters 498:201-203 (2001);Ando et al., Orphanet Journal of Rare Diseases 8:31 (2013); Ruberg andBerk, Circulation 126:1286-1300 (2012); and Johnson et al., J. Mol.Biol. 421(2-3):185-203 (2012). For example, antibodies can be conjugatedto tafamidis, diflunisal, ALN-TTR01, ALNTTR02, ISIS-TTRRx, doxycycline(doxy), tauroursodeoxycholic acid (TUDCA), Doxy-TUDCA, epigallocatechingallate (EGCG), curcumin, or resveratrol (3,5,4′-trihydroxystilbene).Other representative therapeutic moieties include other agents known tobe useful for treatment, management, or amelioration of a TTRamyloidosis or symptoms of a TTR amyloidosis. See, e.g., Ando et al.,Orphanet Journal of Rare Diseases 8:31 (2013) for common clinicalsymptoms of TTR amyloidosis and typical agents used to treat thosesymptoms.

Antibodies can also be coupled with other proteins. For example,antibodies can be coupled with Fynomers. Fynomers are small bindingproteins (e.g., 7 kDa) derived from the human Fyn SH3 domain. They canbe stable and soluble, and they can lack cysteine residues and disulfidebonds. Fynomers can be engineered to bind to target molecules with thesame affinity and specificity as antibodies. They are suitable forcreating multi-specific fusion proteins based on antibodies. Forexample, Fynomers can be fused to N-terminal and/or C-terminal ends ofantibodies to create bi- and tri-specific FynomAbs with differentarchitectures. Fynomers can be selected using Fynomer libraries throughscreening technologies using FACS, Biacore, and cell-based assays thatallow efficient selection of Fynomers with optimal properties. Examplesof Fynomers are disclosed in Grabulovski et al., J. Biol. Chem.282:3196-3204 (2007); Bertschinger et al., Protein Eng. Des. Sel.20:57-68 (2007); Schlatter et al., MAbs. 4:497-508 (2011); Banner etal., Acta. Crystallogr. D. Biol. Crystallogr. 69(Pt6):1124-1137 (2013);and Brack et al., Mol. Cancer Ther. 13:2030-2039 (2014).

The antibodies disclosed herein can also be coupled or conjugated to oneor more other antibodies (e.g., to form antibody heteroconjugates). Suchother antibodies can bind to different epitopes within TTR or a portionthereof or can bind to a different target antigen.

Antibodies can also be coupled with a detectable label. Such antibodiescan be used, for example, for diagnosing a TTR amyloidosis, formonitoring progression of a TTR amyloidosis, and/or for assessingefficacy of treatment. Such antibodies are particularly useful forperforming such determinations in subjects having or being susceptibleto a TTR amyloidosis, or in appropriate biological samples obtained fromsuch subjects. Representative detectable labels that may be coupled orlinked to an antibody include various enzymes, such as horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; prosthetic groups, such streptavidin/biotin andavidin/biotin; fluorescent materials, such as umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;luminescent materials, such as luminol; bioluminescent materials, suchas luciferase, luciferin, and aequorin; radioactive materials, such asyttrium⁹⁰ (90Y), radiosilver-111, radiosilver-199, Bismuth²¹³, iodine(¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I,), carbon (¹⁴C), sulfur (⁵S), tritium (³H),indium (¹¹⁵In, ¹¹³In, ¹¹²In,), technetium (⁹⁹Tc), thallium (²⁰¹Ti),gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon(¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, 159Gd, ¹⁴⁹Pm, 140La, ¹⁷⁵Yb,¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn,⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and ¹¹⁷Tin; positronemitting metals using various positron emission tomographies;nonradioactive paramagnetic metal ions; and molecules that areradiolabelled or conjugated to specific radioisotopes.

Linkage of radioisotopes to antibodies may be performed withconventional bifunction chelates. For radiosilver-111 andradiosilver-199 linkage, sulfur-based linkers may be used. See Hazra etal., Cell Biophys. 24-25:1-7 (1994). Linkage of silver radioisotopes mayinvolve reducing the immunoglobulin with ascorbic acid. Forradioisotopes such as 111In and 90Y, ibritumomab tiuxetan can be usedand will react with such isotopes to form 111In-ibritumomab tiuxetan and90Y-ibritumomab tiuxetan, respectively. See Witzig, Cancer Chemother.Pharmacol., 48 Suppl 1:S91-S95 (2001).

Therapeutic moieties, other proteins, other antibodies, and/ordetectable labels may be coupled or conjugated, directly or indirectlythrough an intermediate (e.g., a linker), to an antibody of theinvention. See e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy,” in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery,” inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review,” in Monoclonal Antibodies 84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy,” inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985); and Thorpe et al., Immunol.Rev., 62:119-58 (1982). Suitable linkers include, for example, cleavableand non-cleavable linkers. Different linkers that release the coupledtherapeutic moieties, proteins, antibodies, and/or detectable labelsunder acidic or reducing conditions, on exposure to specific proteases,or under other defined conditions can be employed.

V. Therapeutic Applications

The above antibodies can be used for treating or effecting prophylaxisof a disease in a patient having or at risk for the disease mediated atleast in part by transthyretin (TTR), and particularly by monomeric,misfolded, aggregated, or fibril forms of TTR. Although an understandingof mechanism is not required for practice, it is believed that any orall of the following mechanisms may contribute to treatment of TTRamyloidosis using the above antibodies: antibody-mediated inhibition ofTTR aggregation and fibril formation, antibody-mediated stabilization ofnon-toxic conformations of TTR (e.g., tetrameric forms), orantibody-mediated clearance of aggregated TTR, oligomeric TTR, ormonomeric TTR. Antibody-drug conjugates can have additional mechanismsof action determined by the conjugated moiety.

Antibodies are administered in an effective regime meaning a dosage,route of administration and frequency of administration that delays theonset, reduces the severity, inhibits further deterioration, and/orameliorates at least one sign or symptom of a disorder being treated. Ifa patient is already suffering from a disorder, the regime can bereferred to as a therapeutically effective regime. If the patient is atelevated risk of the disorder relative to the general population but isnot yet experiencing symptoms, the regime can be referred to as aprophylactically effective regime. In some instances, therapeutic orprophylactic efficacy can be observed in an individual patient relativeto historical controls or past experience in the same patient. In otherinstances, therapeutic or prophylactic efficacy can be demonstrated in apreclinical or clinical trial in a population of treated patientsrelative to a control population of untreated patients.

The frequency of administration depends on the half-life of the antibodyin the circulation, the condition of the patient and the route ofadministration among other factors. The frequency can be daily, weekly,monthly, quarterly, or at irregular intervals in response to changes inthe patient's condition or progression of the disorder being treated. Anexemplary frequency for intravenous administration is between weekly andquarterly over a continuous cause of treatment, although more or lessfrequent dosing is also possible. For subcutaneous administration, anexemplary dosing frequency is daily to monthly, although more or lessfrequent dosing is also possible.

The number of dosages administered depends on whether the disorder isacute or chronic and the response of the disorder to the treatment. Foracute disorders or acute exacerbations of a chronic disorder, between 1and 10 doses are often sufficient. Sometimes a single bolus dose,optionally in divided form, is sufficient for an acute disorder or acuteexacerbation of a chronic disorder. Treatment can be repeated forrecurrence of an acute disorder or acute exacerbation. For chronicdisorders, an antibody can be administered at regular intervals, e.g.,weekly, fortnightly, monthly, quarterly, every six months for at least1, 5 or 10 years, or the life of the patient.

VI. Pharmaceutical Compositions and Methods of Use

Provided herein are several methods of diagnosing, monitoring, treatingor effecting prophylaxis of diseases or conditions mediated at least inpart by transthyretin (TTR), and particularly by monomeric, misfolded,aggregated, or fibril forms of TTR (e.g., TTR amyloidosis). Examples ofsuch diseases include familial TTR amyloidoses, such as familial amyloidcardiomyopathy (FAC) or cardiomyopathy or hypertrophy in athletes orothers undergoing extreme aerobic exercise, familial amyloidpolyneuropathy (FAP), or central nervous system selective amyloidosis(CNSA), and sporadic TTR amyloidoses, such as senile systemicamyloidosis (SSA) or senile cardiac amyloidosis (SCA). TTR amyloidosiscan also be associated as a cause or result of various diseases andconditions characterized by tissue or organ degeneration or trauma.Accumulation of TTR deposits contributes to organ or tissue dysfunctionassociated with the disease or condition. An example of such a conditionamenable to treatment or prophylaxis with the present agents and methodsis spinal stenosis (Westermark et al., Upsala J. Medical Sciences 119,223-238 (2014) and Yanagisawa et al., Modern Pathology 28, 201-207(2015). Another disease likewise amenable to treatment or prophylaxis isosteoarthritis (Takanashi et al., Amyloid 20, 151-155 (2013), Gu et al.,Biomed & Biotechnol. 15, 92-99; Takinami et al., Biomarker Insights 8,85-95 (2014); Akasaki et al., Arthritis Rheumatol. 67, 2097-2107 (2015).Another disease likewise amenable to treatment or prophylaxis isrheumatoid arthritis (Clement et al., JCI Insight 1 epublish (2016).Another disease amenable to treatment or prophylaxis is juvenileidiopathic arthritis (Sharma et al., PLOSone 9, 1-12 (2014). Anotherdisease amenable to treatment or prophylaxis is age related maculardegeneration (wet or dry). Another class of conditions likewise amenableto treatment or prophylaxis are ligament and tendon disorders, such asdisorders of the rotator cuff (Sueyoshi et al., Human Pathol. 42,1259-64 (2011).

Antibodies described above can be incorporated into a pharmaceuticalcomposition for use treatment or prophylaxis of any of the abovediseases and conditions. In general, an antibody or pharmaceuticalcomposition containing an antibody is administered to a subject in needthereof. Patients amenable to treatment include individuals at risk ofTTR amyloidosis but not showing symptoms, as well as patients presentlyshowing symptoms. Some patients can be treated during the prodromalstage of TTR amyloidosis.

Individuals suffering from TTR amyloidosis can sometimes be recognizedfrom the clinical manifestations of TTR amyloidosis, including one ormore of the following: (1) family history of neuropathic disease,especially associated with heart failure; (2) neuropathic pain orprogressive sensory disturbances of unknown etiology; (3) carpal tunnelsyndrome without obvious cause, particularly if it is bilateral andrequires surgical release; (4) gastrointestinal motility disturbances orautonomic nerve dysfunction of unknown etiology (e.g., erectiledysfunction, orthostatic hypotension, neurogenic gladder); (5) cardiacdisease characterized by thickened ventricular walls in the absence ofhypertension; (6) advanced atrio-ventricular block of unknown origin,particularly when accompanied by a thickened heart; and (6) vitreousbody inclusions of the cotton-wool type. See Ando et al., OrphanetJournal of Rare Diseases 8:31 (2013). Definitive diagnosis of TTRamyloidosis, however, typically relies on target organ biopsies,followed by histological staining of the excised tissue with theamyloid-specific dye, Congo red. If a positive test for amyloid isobserved, immunohistochemical staining for TTR is subsequently performedto ensure that the precursor protein responsible for amyloid formationis indeed TTR. For familial forms of the diseases, demonstration of amutation in the gene encoding TTR is then needed before a definitivediagnosis can be made.

The identification of the subject can occur in a clinical setting, orelsewhere, such as in the subject's home, for example, through thesubject's own use of a self-testing kit. For example, the subject can beidentified based on various symptoms such as peripheral neuropathy(sensory and motor), autonomic neuropathy, gastrointestinal impairment,cardiomyopathy, nephropathy, or ocular deposition. See Ando et al.,Orphanet Journal of Rare Diseases 8:31 (2013). The subject can also beidentified by increased levels of non-native forms of TTR in plasmasamples from the subject compared to control samples, as disclosed inthe examples.

As warranted by family history, genetic testing, or medical screeningfor TTR amyloidosis, treatment can begin at any age (e.g., 20, 30, 40,50, 60, or 70 years of age). Treatment typically entails multipledosages over a period of time and can be monitored by assaying antibodyor activated T-cell or B-cell responses to a therapeutic agent (e.g., atruncated form of TTR comprising amino acid residues 89-97) over time.If the response falls, a booster dosage is indicated.

In prophylactic applications, an antibody or a pharmaceuticalcomposition of the same is administered to a subject susceptible to, orotherwise at risk of a disease (e.g., TTR amyloidosis) in a regime(dose, frequency and route of administration) effective to reduce therisk, lessen the severity, or delay the onset of at least one sign orsymptom of the disease. In therapeutic applications, an antibody orimmunogen to induce an antibody is administered to a subject suspectedof, or already suffering from a disease (e.g., TTR amyloidosis) in aregime (dose, frequency and route of administration) effective toameliorate or at least inhibit further deterioration of at least onesign or symptom of the disease.

A regime is considered therapeutically or prophylactically effective ifan individual treated subject achieves an outcome more favorable thanthe mean outcome in a control population of comparable subjects nottreated by methods disclosed herein, or if a more favorable outcome isdemonstrated for a regime in treated subjects versus control subjects ina controlled clinical trial (e.g., a phase II, phase II/III, or phaseIII trial) or an animal model at the p<0.05 or 0.01 or even 0.001 level.

An effective regime of an antibody can be used for, e.g., inhibiting orreducing aggregation of TTR in a subject having or at risk of acondition associated with TTR accumulation; inhibiting or reducing TTRfibril formation in a subject having or at risk of a conditionassociated with TTR accumulation; reducing or clearing TTR deposits oraggregated TTR in a subject having or at risk of a condition associatedwith TTR accumulation; stabilizing non-toxic conformations of TTR in asubject having or at risk of a condition associated with TTRaccumulation; inhibiting toxic effects of TTR aggregates, fibrils ordeposits in a subject having or at risk of a condition associated withTTR accumulation; diagnosing the presence or absence of TTR amyloidaccumulation in a tissue suspected of comprising the amyloidaccumulation; determining a level of TTR deposits in a subject bydetecting the presence of bound antibody in the subject followingadministration of the antibody; detecting the presence of monomeric,misfolded, aggregated, or fibril forms of TTR in a subject; monitoringand evaluating the efficacy of therapeutic agents being used to treatpatients diagnosed with a TTR amyloidosis; inducing an immune responsecomprising antibodies to TTR in a subject; delaying the onset of acondition associated with TTR amyloid accumulation in a subject; ortreating or effecting prophylaxis of a TTR amyloidosis in a patient.

Effective doses vary depending on many different factors, such as meansof administration, target site, physiological state of the subject,whether the subject is human or an animal, other medicationsadministered, and whether treatment is prophylactic or therapeutic.

An exemplary dose range for antibodies can be from about 0.1-20, or0.5-5 mg/kg body weight (e.g., 0.5, 1, 2, 3, 4 or 5 mg/kg) or 10-1500 mgas a fixed dosage. The dosage depends on the condition of the patientand response to prior treatment, if any, whether the treatment isprophylactic or therapeutic and whether the disorder is acute orchronic, among other factors.

Antibody can be administered in such doses daily, on alternative days,weekly, fortnightly, monthly, quarterly, or according to any otherschedule determined by empirical analysis. An exemplary treatmententails administration in multiple doses over a prolonged period, forexample, of at least six months. Additional exemplary treatment regimesentail administration once per every two weeks or once a month or onceevery 3 to 6 months.

Antibodies can be administered via a peripheral route. Routes ofadministration include topical, intravenous, oral, subcutaneous,intraarterial, intracranial, intrathecal, intraperitoneal, intranasal orintramuscular. Routes for administration of antibodies can beintravenous or subcutaneous. Intravenous administration can be, forexample, by infusion over a period such as 30-90 min. This type ofinjection is most typically performed in the arm or leg muscles. In somemethods, agents are injected directly into a particular tissue wheredeposits have accumulated, for example intracranial injection.

Pharmaceutical compositions for parenteral administration can be sterileand substantially isotonic (250-350 mOsm/kg water) and manufacturedunder GMP conditions. Pharmaceutical compositions can be provided inunit dose form (i.e., the dose for a single administration).Pharmaceutical compositions can be formulated using one or morephysiologically acceptable carriers, diluents, excipients orauxiliaries. The formulation depends on the route of administrationchosen. For injection, antibodies can be formulated in aqueoussolutions, e.g., in physiologically compatible buffers such as Hank'ssolution, Ringer's solution, or physiological saline or acetate buffer(to reduce discomfort at the site of injection). The solution cancontain formulatory agents such as suspending, stabilizing and/ordispersing agents. Alternatively antibodies can be in lyophilized formfor constitution with a suitable vehicle, e.g., sterile pyrogen-freewater, before use.

The regimes can be administered in combination with another agenteffective in treatment or prophylaxis of the disease being treated. Suchagents can include siRNA to inhibit expression of TTR or Vyndaqel, astabilizer of TTR in tetramer formation.

After treatment, the subject's condition can be evaluated to determinethe progress or efficacy of such treatment. Such methods preferably testfor changes in TTR amyloid levels or levels of non-native forms of TTR.For example, TTR amyloid levels may be evaluated to determineimprovement relative to the subject's TTR amyloid levels undercomparable circumstances prior to treatment. The subject's TTR amyloidlevels can also be compared with control populations under comparablecircumstances. The control populations can be similarly afflicted,untreated subjects or normal untreated subjects (among other controlsubjects). Improvement relative to similarly afflicted, untreatedsubjects or levels approaching or reaching the levels in untreatednormal subjects indicates a positive response to treatment.

TTR amyloid levels can be measured by a number of methods, includingimaging techniques. Examples of suitable imaging techniques include PETscanning with radiolabeled TTR of fragments thereof, TTR antibodies orfragments thereof, Congo-red-based amyloid imaging agents, such as,e.g., PIB (US 2011/0008255), amyloid-imaging peptide p31(Biodistribution of amyloid-imaging peptide, p31, correlates withamyloid quantitation based on Congo red tissue staining, Wall et al.,Abstract No. 1573, 2011 ISNM Annual Meeting), and other PET labels.Levels of non-native forms of TTR can be measured, for example, byperforming SDS-PAGE/Western blot or Meso Scale Discovery plate assayswith the antibodies disclosed herein on plasma samples or biopsy samplesfrom a subject and comparing to control samples, as described in theexamples.

A. Diagnostics and Monitoring Methods

Also provided are methods of detecting an immune response against TTR ina patient suffering from or susceptible to diseases associated with TTRdeposition or pathogenic forms of TTR (e.g., monomeric, misfolded,aggregated, or fibril forms of TTR). The methods can be used to monitora course of therapeutic and prophylactic treatment with the agentsprovided herein. The antibody profile following passive immunizationtypically shows an immediate peak in antibody concentration followed byan exponential decay. Without a further dose, the decay approachespretreatment levels within a period of days to months depending on thehalf-life of the antibody administered. For example, the half-life ofsome human antibodies is of the order of 20 days.

In some methods, a baseline measurement of antibody to TTR in thesubject is made before administration, a second measurement is made soonthereafter to determine the peak antibody level, and one or more furthermeasurements are made at intervals to monitor decay of antibody levels.When the level of antibody has declined to baseline or a predeterminedpercentage of the peak less baseline (e.g., 50%, 25% or 10%),administration of a further dose of antibody is administered. In somemethods, peak or subsequent measured levels less background are comparedwith reference levels previously determined to constitute a beneficialprophylactic or therapeutic treatment regime in other subjects. If themeasured antibody level is significantly less than a reference level(e.g., less than the mean minus one or, preferably, two standarddeviations of the reference value in a population of subjects benefitingfrom treatment) administration of an additional dose of antibody isindicated.

Also provided are methods of detecting monomeric, misfolded, aggregated,or fibril forms of TTR in a subject, for example, by measuring TTRamyloid or pathogenic forms of TTR (e.g., monomeric, misfolded,aggregated, or fibril forms of TTR) in a sample from a subject or by invivo imaging of TTR in a subject. Such methods are useful to diagnose orconfirm diagnosis of diseases associated with such pathogenic forms ofTTR (e.g., TTR amyloidosis), or susceptibility thereto. The methods canalso be used on asymptomatic subjects. The presence of monomeric,misfolded, aggregated, or fibril forms of TTR indicates susceptibilityto future symptomatic disease. The methods are also useful formonitoring disease progression and/or response to treatment in subjectswho have been previously diagnosed with a TTR amyloidosis.

Biological samples obtained from a subject having, suspected of having,or at risk of having a TTR amyloidosis can be contacted with theantibodies disclosed herein to assess the presence of monomeric,misfolded, aggregated, or fibril forms of TTR. For example, levels ofmonomeric, misfolded, aggregated, or fibril forms of TTR in suchsubjects may be compared to those present in healthy subjects.Alternatively, levels of TTR amyloid or pathogenic forms of TTR (e.g.,monomeric, misfolded, aggregated, or fibril forms of TTR) in suchsubjects receiving treatment for the disease may be compared to those ofsubjects who have not been treated for a TTR amyloidosis. Some suchtests involve a biopsy of tissue obtained from such subjects. ELISAassays may also be useful methods, for example, for assessing levels ofmonomeric, misfolded, aggregated, or fibril forms of TTR in fluidsamples. Some such ELISA assays involve anti-TTR antibodies thatpreferentially bind monomeric, misfolded, aggregated, or fibril forms ofTTR relative to normal tetrameric forms of TTR.

The in vivo imaging methods can work by administering a reagent, such asantibody that binds to monomeric, misfolded, aggregated, or fibril formsof TTR in the subject, and then detecting the reagent after it hasbound. Such antibodies typically bind to an epitope within residues89-97 of TTR. If desired, the clearing response can be avoided by usingantibody fragments lacking a full length constant region, such as Fabs.In some methods, the same antibody can serve as both a treatment anddiagnostic reagent.

Diagnostic reagents can be administered by intravenous injection intothe body of the subject, or via other routes deemed reasonable. The doseof reagent should be within the same ranges as for treatment methods.Typically, the reagent is labeled, although in some methods, the primaryreagent with affinity for monomeric, misfolded, aggregated, or fibrilforms of TTR is unlabeled and a secondary labeling agent is used to bindto the primary reagent. The choice of label depends on the means ofdetection. For example, a fluorescent label is suitable for opticaldetection. Use of paramagnetic labels is suitable for tomographicdetection without surgical intervention. Radioactive labels can also bedetected using PET or SPECT.

Diagnosis is performed by comparing the number, size, and/or intensityof labeled loci to corresponding base line values. The base line valuescan represent the mean levels in a population of undiseased individuals.Base line values can also represent previous levels determined in thesame subject. For example, base line values can be determined in asubject before beginning treatment, and measured values thereaftercompared with the base line values. A decrease in values relative tobase line generally signals a positive response to treatment.

IX. Kits

The invention further provides kits (e.g., containers) comprising anantibody disclosed herein and related materials, such as instructionsfor use (e.g., package insert). The instructions for use may contain,for example, instructions for administration of the antibody andoptionally one or more additional agents. The containers of antibody maybe unit doses, bulk packages (e.g., multi-dose packages), or sub-unitdoses.

Package insert refers to instructions customarily included in commercialpackages of therapeutic products that contain information about theindications, usage, dosage, administration, contraindications and/orwarnings concerning the use of such therapeutic products

Kits can also include a second container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It can also include other materials desirable from acommercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

X. Other Applications

The antibodies can be used for detecting monomeric, misfolded,aggregated, or fibril forms of transthyretin (TTR), or fragmentsthereof, in the context of clinical diagnosis or treatment or inresearch. For example, the antibodies can be used to detect the presenceof monomeric, misfolded, aggregated, or fibril forms of TTR in abiological sample as an indication that the biological sample comprisesTTR amyloid deposits. Binding of the antibodies to the biological samplecan be compared to binding of the antibodies to a control sample. Thecontrol sample and the biological sample can comprise cells of the sametissue origin. Control samples and biological samples can be obtainedfrom the same individual or different individuals and on the sameoccasion or on different occasions. If desired, multiple biologicalsamples and multiple control samples are evaluated on multiple occasionsto protect against random variation independent of the differencesbetween the samples. A direct comparison can then be made between thebiological sample(s) and the control sample(s) to determine whetherantibody binding (i.e., the presence of monomeric, misfolded,aggregated, or fibril forms of TTR) to the biological sample(s) isincreased, decreased, or the same relative to antibody binding to thecontrol sample(s). Increased binding of the antibody to the biologicalsample(s) relative to the control sample(s) indicates the presence ofmonomeric, misfolded, aggregated, or fibril forms of TTR in thebiological sample(s). In some instances, the increased antibody bindingis statistically significant. Optionally, antibody binding to thebiological sample is at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold,10-fold, 20-fold, or 100-fold higher than antibody binding to thecontrol sample.

In addition, the antibodies can be used to detect the presence ofmonomeric, misfolded, aggregated, or fibril forms of TTR in a biologicalsample to monitor and evaluate the efficacy of a therapeutic agent beingused to treat a patient diagnosed with a TTR amyloidosis. A biologicalsample from a patient diagnosed with a TTR amyloidosis is evaluated toestablish a baseline for the binding of the antibodies to the sample(i.e., a baseline for the presence of the monomeric, misfolded,aggregated, or fibril forms of TTR in the sample) before commencingtherapy with the therapeutic agent. In some instances, multiplebiological samples from the patient are evaluated on multiple occasionsto establish both a baseline and measure of random variation independentof treatment. A therapeutic agent is then administered in a regime. Theregime may include multiple administrations of the agent over a periodof time. Optionally, binding of the antibodies (i.e., presence ofmonomeric, misfolded, aggregated, or fibril forms of TTR) is evaluatedon multiple occasions in multiple biological samples from the patient,both to establish a measure of random variation and to show a trend inresponse to immunotherapy. The various assessments of antibody bindingto the biological samples are then compared. If only two assessments aremade, a direct comparison can be made between the two assessments todetermine whether antibody binding (i.e., presence of monomeric,misfolded, aggregated, or fibril forms of TTR) has increased, decreased,or remained the same between the two assessments. If more than twomeasurements are made, the measurements can be analyzed as a time coursestarting before treatment with the therapeutic agent and proceedingthrough the course of therapy. In patients for whom antibody binding tobiological samples has decreased (i.e., the presence of monomeric,misfolded, aggregated, or fibril forms of TTR), it can be concluded thatthe therapeutic agent was effective in treating the TTR amyloidosis inthe patient. The decrease in antibody binding can be statisticallysignificant. Optionally, binding decreases by at least 1%, 2%, 3%, 4%,5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.Assessment of antibody binding can be made in conjunction with assessingother signs and symptoms of TTR amyloidosis.

The antibodies can also be used as research reagents for laboratoryresearch in detecting monomeric, misfolded, aggregated, or fibril formsof TTR, or fragments thereof. In such uses, antibodies can be labeledwith fluorescent molecules, spin-labeled molecules, enzymes, orradioisotopes, and can be provided in the form of kit with all thenecessary reagents to perform the detection assay. The antibodies canalso be used to purify monomeric, misfolded, aggregated, or fibril formsof TTR, or binding partners of monomeric, misfolded, aggregated, orfibril forms of TTR, e.g., by affinity chromatography.

Antibody 9D5 has been deposited subject to the Budapest Treaty underaccession number PTA-124078 on Apr. 4, 2017 at the American Type CultureCollection 10801 University Boulevard Manassas, Va. 20110 USA. Allrestrictions imposed by the depositor on the availability to the publicof the deposited material will be irrevocably removed on the granting ofa patent except as permitted under 37 CFR 1.808(b).

Antibody 14G8 has been deposited subject to the Budapest Treaty underaccession number PTA-124079 on Apr. 4, 2017 at the American Type CultureCollection 10801 University Boulevard Manassas, Va. 20110 USA. Allrestrictions imposed by the depositor on the availability to the publicof the deposited material will be irrevocably removed on the granting ofa patent except as permitted under 37 CFR 1.808(b).

The antibodies can also be used for inhibiting or reducing aggregationof TTR, inhibiting or reducing TTR fibril formation, reducing orclearing TTR deposits or TTR aggregates, or stabilizing non-toxicconformations of TTR in a biological sample. The biological sample cancomprise, for example, blood, serum, plasma, or tissue (e.g., tissuefrom the heart, peripheral nervous system, autonomic nervous system,kidneys, eyes, or gastrointestinal tract). In some instances, TTRaggregation, TTR fibril formation, or TTR deposits are inhibited orreduced by at least 10%, 20%, 25%, 30%, 40%, 50%, or 75%, (e.g., 10%-75%or 30%-70%). Assays for detecting fibril formation are describedelsewhere herein. See also US 2014/0056904.

All patent filings, web sites, other publications, accession numbers andthe like cited above or below are incorporated by reference in theirentirety for all purposes to the same extent as if each individual itemwere specifically and individually indicated to be so incorporated byreference. If different versions of a sequence are associated with anaccession number at different times, the version associated with theaccession number at the effective filing date of this application ismeant. The effective filing date means the earlier of the actual filingdate or filing date of a priority application referring to the accessionnumber if applicable. Likewise if different versions of a publication,website or the like are published at different times, the version mostrecently published at the effective filing date of the application ismeant unless otherwise indicated. Any feature, step, element,embodiment, or aspect of the invention can be used in combination withany other unless specifically indicated otherwise. Although the presentinvention has been described in some detail by way of illustration andexample for purposes of clarity and understanding, it will be apparentthat certain changes and modifications may be practiced within the scopeof the appended claims.

EXAMPLES Example 1. Identification of mis-TTR Monoclonal Antibodies

Conformationally-specific monoclonal antibodies against monomeric,mis-folded, fibril, or aggregated forms of TTR (mis-TTR) were generated,screened, expressed, and purified as described in Materials and Methods(a-d). In order to generate mis-TTR monoclonal antibodies, the crystalstructure of human tetrameric TTR was examined to find regions of theprotein that are buried in the tetramer, but exposed upon dissociationof the tetramer into its monomeric subunits. The region identified wasresidues 89-97 (EHAEVVFTA) (SEQ ID NO: 113) located within the F strandof TTR and sequestered at the dimer interface of the tetrameric protein.A BLAST search of the protein database did not reveal any other humanproteins possessing this sequence.

A peptide comprising this sequence (ggEHAEVVFTAggkg) (SEQ ID NO: 114),was synthesized. Capitalized letters represent residues 89-97 of TTR.Lower case letters represent additional linker residues added toincrease the solubility of the antigenic peptide and to establish the 9amino acid fragment as an internal sequence. This peptide was linked toa poly-lysine dendritic core, generating a multiple antigenic peptideimmunogen (TTR-MAP) comprising a core of lysine residues with multiplebranches linked to the TTR 89-97 peptide. The antibodies listed in Table2 were generated against TTR-MAP.

In addition to this multiple antigenic peptide, two other immunogenscontaining the same TTR fragment were generated by covalently linkingsimilar TTR 89-97 peptides (Ac-cggEHAEVVFTA-amide (SEQ ID NO: 115) andAc-EHAEVVFTAcgg-amide (SEQ ID NO: 116)) via the N- and C-terminalcysteine residues to keyhole limpet hemocyanin (TTR89-97-N-KLH andTTR89-97-C-KLH).

Following antibody generation, screening, expression, and purification,detailed binding kinetic parameters (association rate (k_(a)),dissociation rate (k_(d)), and binding affinity constant (K_(D))) weredetermined for lead mis-TTR antibodies by Surface Plasmon Resonance(SPR) for recombinant human TTR F87M/L110M, as shown in Table 2.Anti-mouse IgG (GE Healthcare) was immobilized on a sensor chip C5(lacking dextran chains) via amine coupling following the instructionsprovided in the GE Healthcare anti-mouse kit, and mis-TTR mAbs werecaptured to a level to ensure a maximum binding of analyte of 30-50 RU.Various concentrations of analyte (recombinant human TTR F87M/L110M)were passed over the captured ligand at 30 μl/min in running buffer(HBS+0.05% P-20, 1 mg/mL BSA) in 3-fold dilutions. For eachconcentration, the reaction proceeded for a time frame allowing for thehigher analyte concentrations to reach equilibrium during association,as well as at least 10% of signal to decay during dissociation. At leastone concentration (not the highest or lowest) was run in duplicate.Concentration ranges of analyte were selected based on preliminaryexperimentation to span at least 10-fold above K_(D) to 10-fold belowK_(D).

The results of SPR analysis of lead mis-TTR mAbs is shown in Table 2below.

TABLE 2 SPR Analysis of Lead mis-TTR Antibodies Binding to Human TTR(F87M/L110M) mAb k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) R_(max) 9D5 2.715E+44.930E−4 1.816E−8 31.55 14G8 2.880E+4 5.358E−4 1.861E−8 27.13 5A16.107E+4 4.693E−4 7.684E−9 30.98 6C1 4.607E+4 4.151E−4 9.010E−9 26.32

Example 2. Binding of mis-TTR Antibodies to TTR Antigen

Four lead mis-TTR mAbs (9D5, 14G8, 6C1, and 5A1) were assayed by ELISAat concentrations ranging from 0.31 to 2.5 μg/ml using bothpH4.0-treated TTR (pH4-TTR) and native TTR as the coating antigen. TTRantigen preparation and ELISA protocols are described elsewhere inMaterials and Methods (e-g).

The resulting binding curves and tabulated K_(d) and B_(max) values areshown in FIG. 3 and Table 3 below. The results in FIG. 3 are presentedin arbitrary units (a.u.) on the y-axis. All mAbs showed significantbinding to pH4-TTR with K_(d) values ranging from 16 nM (6C1) to 282 nM(9D5). B_(max) values for binding to pH4-TTR ranged from a low of 0.65a.u. (14G8) to a high of 2.02 (9D5). In contrast to the binding topH4-TTR, none of the antibodies showed significant binding to nativeTTR, indicating that all TTR antibodies generated were specific fornon-native forms of TTR.

TABLE 3 ELISA Analysis of Lead mis-TTR Antibodies Binding to pH4-TTR mAbK_(d) (nM) B_(max) (a.u.) 9D5 282 2.02 14G8 108 0.65 6C1 16 1.07 5A1 231.61

Example 3. Analysis of mis-TTR Antibodies by SDS-PAGE and Native-PAGE

9D5 and 14G8 were analyzed by SDS-PAGE/Western to demonstratespecificity of binding toward monomeric/denatured forms of TTR versusnative, non-denatured TTR. SDS-PAGE, Native-PAGE, and Western Blotprotocols are described elsewhere in the Methods and Materials (h-j).

Non-denatured TTR or pH4-TTR was run on an SDS-PAGE gel alongsideheat-denatured TTR and heat-denatured pH4-TTR. After electrophoresis,the gel was Western blotted onto nitrocellulose and stained with TTRmAbs 9D5 and 14G8. Both antibodies only recognized TTR when it wastreated at pH4 or when TTR or pH4-TTR was first heat-denatured prior toSDS-PAGE. These 9D5 and 14G8 thus show a specificity for TTR conformersgenerated either by denaturation of TTR or by treatment of TTR at pH4.

6C1 and 5A1 along with total TTR mAbs (7G7, 8C3) and the commerciallyavailable Sigma polyclonal antibody were also analyzed bySDS-PAGE/Western. Each blot contained stained molecular weight markers,non-denatured TTR, and pH4-TTR.

The stained SDS-PAGE gel showed that the major species present in thenon-denatured TTR sample was an ˜38 kDa dimer. In contrast, the majorcomponent present in the pH4-TTR sample ran as an ˜35 kDa dimer with asmall amount of dimer of an ˜15 kDa monomer. This dimer ran as aslightly smaller protein than the dimer present in the non-denatured TTRsample, indicating a conformational difference between these two TTRdimer species.

The Western blots of TTR and pH4-TTR using the four mis-TTR antibodiesshowed that these mAbs do not recognize non-denatured TTR, but do bindto both the denatured monomer and dimer present in the pH4-TTR sample.Thus, the four mis-TTR mAbs (9D5, 14G8, 6C1, and 5A1) show similarspecificities for non-native conformations of TTR when analyzed bySDS-PAGE/Westerns.

In contrast to the four mis-TTR mAbs, the two TTR control mAbs, 7G7 and8C3, generated through immunization of mice with intact TTR recognizedall TTR species present in the TTR and pH4-TTR samples, includingtetrameric TTR species. Thus unlike the mis-TTR mAbs, these control mAbsbind TTR but with no conformational specificity. The Sigma polyclonalantibody behaved similarly to the 7G7 and 8C3 control mAbs.

TTR and pH4-TTR were also run on a native gel to see if the four mis-TTRmAbs were capable of showing conformation specificity undernon-denaturing gel conditions. On a stained native PAGE gel, TTR ran asan ˜35 kDa native dimer with a small amount of tetramer. In contrast,pH4-TTR ran primarily as a high molecular-weight smear with a traceamount of the ˜35 kDa dimer. The non-specific Sigma polyclonal antibodyrecognized all TTR species present in both the TTR and the pH4-TTRsample. In contrast, 9D5 only recognized the high molecular weight TTRspecies present in the pH4-TTR sample. As observed in theSDS-PAGE/Western study, 9D5 did not recognize any of the native TTRspecies.

All four mis-TTR mAbs were subsequently analyzed by native-PAGE/Westernblot. As expected and similar to 9D5, the other mis-TTR mAbs, 14G8, 6C1,and 5A1, specifically bound to the high molecular weight non-nativeforms of TTR present in the pH4-TTR sample. None of these antibodiesrecognized the ˜35 kDa native TTR dimer. These results indicate that thefour mis-TTR mAbs behave similarly and recognize only non-native TTRspecies that are conformationally distinct from native TTR.

Example 4. Inhibition of TTR Fiber Formation by mis-TTR Antibodies

TTR-Y78F is a TTR variant containing a point mutation at position 78 inthe protein sequence that destabilizes the TTR tetramer. With time andunder mildly acidic conditions, this TTR variant dissociates into itsmonomeric subunits which can then go on to aggregate and form fiberscapable of binding to thioflavin-T. The extent of fiber formation canthus be monitored by measuring thioflavin-T fluorescence at 480 nm.Introduction of a mis-TTR antibody specific for dissociated TTR monomersor aggregates would prevent the assembly of TTR fibers resulting in adecrease in thioflavin-T fluorescence relative to a no-antibody controlreaction. Protocols for examining inhibition of TTR fiber formation aredescribed elsewhere in the Materials and Methods (k).

All four mis-TTR antibodies strongly inhibited the formation ofthioflavin-T reactive TTR-Y78F fibers relative to the isotype control.The results are shown in FIG. 4A and are presented in arbitrary units(a.u.) on the y-axis. Mis-TTR antibody 5A1 almost completely inhibitedfiber formation. These results are consistent with the notion thatmis-TTR antibodies bind monomeric and/or aggregated forms of TTR,thereby preventing the formation of TTR fibers.

Table 4 summarizes the characterization data obtained for the set of 4mis-TTR antibodies (9D5, 14G8, 6C1, and 5A1) that showed goodconformational selectivity for non-native forms of TTR. These antibodieshad affinities (K_(D)) for pH4-TTR ranging from 14.5 nM (6C1) to 257 nM(9D5) and B_(max) values ranging from 0.65 a.u. (14G8) to 2.02 (9D5).None of these antibodies recognized native TTR, but did bind to pH4-TTRon an SDS-PAGE/Western and to the high molecular weight TTR aggregateson a native-PAGE/Western. These antibodies also inhibited the formationof TTR fibrils in the fibril formation assay using Thio-T as theread-out.

TABLE 4 mis-TTR-Y78F mAb Characterization Summary Table Sandwich ELISA(pH 4-TTR) Western Blot K_(D) B_(max) SDS-PAGE Native % Inh. FibrilsClone ID (nM) (OD₄₅₀ a.u.) (TTR) (pH 4-TTR) (HMW-TTR) (Thio-T) 9D5 2572.02 − +++ +++ 83 14G8 98.7 0.65 − +++ ++ 65 6C1 14.6 1.07 − +++ +++ 725A1 21.3 1.61 − +++ +++ 100

TTR-V122I is a TTR variant containing a single point mutation atposition 122 that destabilizes the tetramer. Fibril formation wasassociated with an increase in ThT fluorescence Increasing 14G8 mAbconcentrations caused a monotonic decrease in ThT fluorescenceindicating a substoichiometric inhibition of TTR fibrillation(IC₅₀=0.028±0.009 mg/mL; n=3; FIG. 4B and Table 4a). The isotype controlmAb did not cause inhibition of TTR fibrillation (FIG. 4C), thusdemonstrating the specificity of 14G8 mediated inhibition.

Comparable substoichiometric IC50 values determined for 5A1 and 6C1(Table 4a) suggested analogous mechanisms of fibril inhibition for eachof these mis-TTR mAbs. In contrast, 9D5 unexpectedly failed to inhibitTTR-V122I fibril formation, despite showing similar specificity andaffinity for non-native TTR. It remains to be explored whether 9D5 ismore sensitive to the assay conditions used.

TABLE 4a mis-TTR-V122I mAb Characterization Summary Table Antibody IC₅₀± SD (mg/mL) 9D5 No inhibition 14G8 0.028 ± 0.009 6C1 0.048 ± 0.059 5A10.015 ± 0.02  EG 27/1 No inhibition

Example 5. Immunohistochemical (IHC) Characterization of ATTR TissueUsing mis-TTR mAbs

The lead mis-TTR mAbs raised to the TTR 89-97 fragment of thetransthyretin protein were immunohistochemically tested on fresh frozenand paraffin processed tissue from confirmed TTR cardiac amyloidosispatients. Protocols for obtaining and preparing cardiac tissue samples,immunohistochemistry (IHC), and image analysis, are provided elsewherein the Materials and Methods (l-o). The antibodies used for IHC aredescribed in Table 5.

TABLE 5 Antibodies Used for Immunohistochemical CharacterizationAntibody Stain Cardiac Concen- Antibody Type Vendor Tissue tration 14G8mis-TTR Prothena Yes 0.5 μg/mL Biosciences 9D5 mis-TTR Prothena Yes 0.5μg/mL Biosciences 6C1 mis-TTR Prothena Yes 0.5 μg/mL Biosciences 5A1mis-TTR Prothena Yes 0.5 μg/mL Biosciences 7G7 TTR Prothena Yes 0.5μg/mL Biosciences 6F10 Isotype Prothena No 0.5 μg/mL Control BiosciencesPrealbumin TTR Dako North Yes 1:2,000 & (A0002) America 1:20,000 KappaLight LC-κ Dako North No 1:8,000 Chains America (A0191) Lambda LightLC-λ Dako North No 1:8,000 Chains America (A0193) Amyloid A AA DakoNorth No 1:8,000 (M0759) America

Cardiac tissue samples were obtained from patients with confirmeddiagnoses of ATTR mutations. Demographics for cases examinedimmunohistochemically were as follows and are summarized in Table 6:FAC=familial amyloidotic cardiomyopathy; FAP=familial amyloidoticpolyneuropathy; 1° AL=light-chain amyloidosis;ATTR=transthyretin-mediated amyloidosis; Unk=Unknown

TABLE 6 Immunohistochemical Staining of Cardiac Tissue Samples withmis-TTR Antibodies Stained with TTR TTR Case Diagnosis Mutations FormatAntibodies? Patient 1 FAC Ileu122 Frozen Yes Patient 2 FAP Wild typeFrozen Yes Patient 3 FAP 84Ser Frozen Yes Patient 4 FAP 84Ser Frozen YesPatient 5 1° AL — Frozen No Patient 6 1° AL — Frozen No Patient 7 ATTR10Arg Frozen Yes Patient 8 ATTR V122I Frozen Yes Patient H1 ATTRVal122Ile FFPE Yes Patient H2 ATTR Thr60Ala FFPE Yes Patient H3 ATTRThr49Ala FFPE Yes Patient H4 ATTR Ile84Ser FFPE Yes Patient H5 Unk.Senile Cardiac FFPE Yes Patient H6 ATTR Ile84Ser FFPE Yes

Mouse monoclonal antibodies (mis-TTR mAbs) raised to the 89-97 fragmentof the transthyretin protein were immunohistochemically tested on freshfrozen and paraffin processed tissue from confirmed TTR cardiacamyloidosis patients. Each mis-TTR antibody showed strongimmunoreactivity on ATTR cardiac tissue. Dark staining was observed indeposits throughout the myocardium and the vasculature. Whenimmunoreactivity was compared to staining with Congo Red ofThioflavin-T, the majority of the immunoreactivity in the tissue showedhigh congruence with Congo red birefringence and Thioflavin T-positivestaining. This confirms the beta pleated sheet nature of the TTR amyloiddeposited in this tissue. 14G8, 9D5, 6C1, and 5A1 also detectedpre-amyloid TTR present in areas of the myocardium that wereTTR-immunopositive but Congo red or Thioflavin T-negative. Both theIgG-isotype control antibody and primary antibody omission sections werenegative for staining across all tissues tested. Antibodies reactivetoward other amyloidogenic proteins (lambda and kappa light chains oramyloid A) were non-reactive toward the ATTR cardiac tissue used in thisanalysis, indicating that deposits were specifically TTR in nature.

The staining patterns of mis-TTR antibodies were compared to thatobtained with a well-characterized commercial TTR reference antibody(prealbumin, A0002; Dako; Carpinteria, Calif.). The DAKO referenceantibody stained the diseased myocardium in the same areas as themis-TTR antibodies, but produced a more diffuse staining pattern. TheDAKO reference antibody did not stain the congophillic TTR amyloiddeposits present on the vasculature as strongly as the mis-TTRantibodies.

The mis-TTR antibodies did not stain normal, non-disease tissue.Furthermore, as expected, staining with an isotype control antibody,6F10, was also negative.

In order to determine if the reactivity of mis-TTR antibodies wasspecific for TTR deposits, cross reactivity of these antibodies towardcardiac tissue derived from patients diagnosed with primary ALamyloidosis was examined. As expected, no staining of AL amyloid tissuewas observed, confirming that TTR antibodies react specifically towardATTR diseased tissue.

Cardiac tissue from patients with confirmed diagnoses of senile systemicamyloidosis or from patients with confirmed FAC, or FAP caused by pointmutations in the TTR gene also stained positively with 14G8, 9D5, 6C1,and 5A1. These results indicate that mis-TTR antibodies have the abilityto recognize TTR deposits in cardiac tissue regardless of the ATTRgenotype.

Other non-cardiac tissues known to express TTR were also examined forstaining by mis-TTR antibodies and compared to the staining obtainedusing the DAKO reference antibody. As expected, the liver, pancreas andchoroid plexus all stained positively for TTR using the Dako referenceantibody. In contrast, 14G8 only stained the pancreatic alpha cellslocated in the islets of Langerhans and the choroid plexus, suggestingthat some of the TTR localized to these organs are conformationallydistinct from TTR expressed in the liver. The lack of mis-TTR mAbimmunoreactivity in the liver suggests that the large amount of TTRexpressed there is primarily tetrameric, native TTR and does not havethe exposed mis-TTR epitope. Similar results were obtained when the sametissues were stained with 9D5, 6C1, and 5A1.

Example 6. Analysis of ATTR vs Normal Human Plasma by SDS-PAGE/WesternBlot and by Meso Scale Discovery (MSD) Plate Assay

Six plasma samples from patients confirmed for V30M ATTR (Sample #11,#12, #15, #18, #19, #20) and 6 samples from normal subjects (#21, #22,#23, #24, #25, #27) were obtained from M. Saraiva (Porto University,Portugal). Sample # C6 was a normal human serum sample obtained from acommercial source (BioreclamationIVT). Samples were analyzed by SDS-PAGEand Western blot, or by MesoScale Discovery (MSD) Plate Assay. Protocolsfor these assays are described elsewhere in the Materials and Methods(p-r). A standard curve was generated for the MSD Plate Assay using 6C1.

In the resulting Western blots using the 9D5 and the 5A1 mis-TTR mAbs,differences in banding patterns between normal and TTR-V30M diseasedplasma samples could be detected. All plasma samples contained an ˜14kDa TTR band that co-migrated with the non-native TTR monomer present inthe pH4-TTR reference sample. In general, plasma samples derived fromTTR-V30M patients (#21, 22, 23, 24, 25, & 27) had more of this monomericmis-TTR species. In addition, plasma samples derived from V30M patientsalso contained an ˜30 kDa band that co-migrated with the non-native TTRdimer present in the pH4-TTR reference sample. With the exception ofsamples #12 and #18, plasma samples derived from normal individualspossessed less of this dimer species.

The above Western blots were scanned and the intensities of the combined9D5- or 5A1-reactive TTR dimer and monomer bands were plotted for eachsample. The results are shown in FIGS. 5A (9D5) and 5B (5A1) and arepresented in arbitrary units (a.u.) on the y-axis. With the exception ofplasma samples #15 and #18, plasma samples derived from normalindividuals (11, 12, 19, and 20) contained less 9D5- and 5A1-reactivedimer and monomer species than samples derived from V30M patients (21-25and 27).

The 12 serum samples analyzed by 9D5 and 5A1 Western blot were alsoanalyzed by MSD plate assay using 6C1 as the mis-TTR capture antibodyand the Dako-SulfoTag antibody as the detection antibody. Results ofthese MSD assays are shown in FIG. 6 and are presented in arbitraryunits (a.u.) on the y-axis. Samples 11, 12, 15, 18, 19, and 20 representnormal plasma. Samples 21-25 and 27 represent V30M diseased plasma.

With the exception of plasma samples #15 and #18, the amount of6C1-reactive TTR present in plasma samples derived from normalindividuals was lower than that observed in plasma from TTR-V30Mdiseased individuals. The levels of 6C1 reactivity measured by MSD assaycorrelated very well with the amount of 9D5 reactive dimer and monomerobserved above by SDS-PAGE/Western.

In order to determine the concentration of the reactive TTR speciespresent in plasma samples, the same samples were re-assayed using 6C1 asthe capture antibody and 8C3-SulfoTag as the detection antibody. MSDsignals were converted to ng/ml concentrations of reactive TTR speciesusing the TTR F87M/L110M standard curve generated above. Based on thisanalysis, the average concentration of 6C1-reactive TTR present in thecontrol samples was 271+/−185 ng/ml. In contrast, the averageconcentration of reactive TTR present in the V30M diseased plasmasamples was higher, at 331+/−95 ng/ml. Taken together, these MSD resultssuggest that mis-TTR antibodies are capable of distinguishing betweenATTR disease versus normal plasma. This warrants further development ofmis-TTR antibodies for use in diagnostic assays of ATTR disease.

Example 7. Design of Humanized 9D5 Antibodies

The starting point or donor antibody for humanization was the mouseantibody 9D5. The heavy chain variable amino acid sequence of maturem9D5 is provided as SEQ ID NO: 1. The light chain variable amino acidsequence of mature m9D5 is provided as SEQ ID NO: 16. The heavy chainCDR1, CDR2, and CDR3 amino acid sequences are provided as SEQ ID NOS:13-15, respectively (as defined by Kabat). A composite Chothia-KabatCDR-H1 is provided as SEQ ID NO: 117. The light chain CDR1, CDR2, andCDR3 amino acid sequences are provided as SEQ ID NOS: 24-26,respectively (as defined by Kabat). Kabat numbering is used throughoutin this Example.

The variable kappa (Vk) of m9D5 belongs to mouse Kabat subgroup 2, whichcorresponds to human Kabat subgroup 3. The variable heavy (Vh) of m9D5belongs to mouse Kabat subgroup 3d, which corresponds to Kabat subgroup3. See Kabat et al. Sequences of Proteins of Immunological Interest,Fifth Edition. NIH Publication No. 91-3242, 1991. The 16-residue CDR-L1belongs to canonical class 4, the 7-residue CDR-L2 belongs to canonicalclass 1, and the 9-residue CDR-L3 belongs to canonical class 1 in Vk.See Martin & Thornton, J. Mol. Biol. 263:800-15, 1996. The 10-residuecomposite Chothia-Kabat CDR-H1 belongs to canonical class 1, and the17-residue CDR-H2 belongs to canonical class 1. See Martin & Thornton, JMol. Biol. 263:800-15, 1996. The CDR-H3 has no canonical classes.

The residues at the interface between the Vk and Vh domains are the onescommonly found, except that Leu is at position 47 in heavy chain,whereas Tyr typically is that this position. This position is acandidate for backmutation.

A search was made over the protein sequences in the PDB database(Deshpande et al., Nucleic Acids Res. 33: D233-7, 2005) to findstructures which would provide a rough structural model of 9D5. Thecrystal structure of antibody fab (pdb code 1MJU) (Ruzheinikov et al.,J. Mol. Biol. 332(2):423-435, 2003) was used for the Vk structure sinceit had good resolution (1.22 A) and overall sequence similarity to 9D5Vk, retaining the same canonical structure for the loop as 9D5. Amonomeric antibody with pdb code 1SEQ (Covaceuszach et al., ActaCrystallogr. D Biol. Crystallogr. 57 (PT 9), 1307-1309, 2001) was usedfor the Vh structure since it had good sequence similarity andresolution (2.0 A), and it has the same canonical structures for CDR-H1and CDR-H2 as that of 9D5 VH. We modeled 9D5 Vh chain using the 1MQKstructure as well, since it has a better resolution of 1.28 A(Ostermeier et al., Proteins 21(1):74-77, 1995). BioLuminate software(licensed from Schrodinger Inc.) was used to model a rough structure of9D5.

A search of the non-redundant protein sequence database from NCBIallowed selection of suitable human frameworks into which to graft themurine CDRs. For Vh, human Ig heavy chain BAC02114 (GI: 21670209) (SEQID NO: 3) was chosen (Akahori et al., Construction and characterizationof antibody libraries: isolation of therapeutic human antibodies andapplication to functional genomics, Direct Submission, Jul. 25, 2001).It shares the canonical forms of 9D5. It is a member of Kabat humanheavy subgroup 1. 9D5 Vh has some unique framework residue and any humanframework acceptor did not show very high homology. Therefore, we used asecond Framework, AAX82494 (GI: 62421461) (SEQ ID NO: 4) (Lundquist etal., Infect. Immun. 74(6), 3222-3231, 2006) as well to make a hybridacceptor framework. For Vk, a human kappa light chain with NCBIaccession code ABC66952 (GI: 84798006) (Shriner et al., Vaccine24(49-50):7159-7166, 2006) was chosen (SEQ ID NO: 18). It has the samecanonical classes for CDR-L1 and L2 as that for the parental Vk. It is amember of Kabat human kappa subgroup 2.

Eight humanized heavy chain variable region variants and five humanizedlight chain variable region variants were constructed containingdifferent permutations of substitutions (Hu9D5VHv1, 2, 2b, 3, 3b, 4, 4b,and 5 (SEQ ID NOS: 5-12, respectively) and Hu9D5VLv1-5 (SEQ ID NOS:19-23, respectively)) (FIGS. 12A-E and FIGS. 13A-D). The exemplaryhumanized Vh and Vk designs, with backmutations and other mutationsbased on selected human frameworks, are shown in FIGS. 12A-E and FIGS.13A-D, respectively. The gray-shaded areas in the first column in FIGS.12A-E and FIGS. 13A-D indicate the CDRs as defined by Chothia, and thegray-shaded areas in the remaining columns in FIGS. 12A-E and FIGS.13A-D indicate the CDRs as defined by Kabat. SEQ ID NOS: 5-12 and 19-23contain backmutations and other mutations as shown in Table 7. The aminoacids at positions H42, H47, H69, H82, H82(b), H108, L8, L9, L18, L19,L36, L39, L60, L70, and L74 in Hu9D5VHv1, 2, 2b, 3, 3b, 4, 4b, and 5 andHu9D5VLv1-5 are listed in Tables 8 and 9.

TABLE 7 V_(H), V_(L) Backmutations and Other Mutations V_(H) or V_(L)Variant V_(H) or V_(L) Exon Acceptor Sequence Donor Framework ResiduesHu9D5VHv1 NCBI accession codes BAC02114 and H47, H69, H82 (SEQ ID NO: 5)AAX82494 (SEQ ID NOS: 3 and 4, respectively) Hu9D5VHv2 NCBI accessioncodes BAC02114 and H47, H69, H82, H82b (SEQ ID NO: 6) AAX82494 (SEQ IDNOS: 3 and 4, respectively) Hu9D5VHv2b NCBI accession codes BAC02114 andH42, H47, H108 (SEQ ID NO: 7) AAX82494 (SEQ ID NOS: 3 and 4,respectively) Hu9D5VHv3 NCBI accession codes BAC02114 and H69, H82, H82b(SEQ ID NO: 8) AAX82494 (SEQ ID NOS: 3 and 4, respectively) Hu9D5VHv3bNCBI accession codes BAC02114 and H47, H108 (SEQ ID NO: 9) AAX82494 (SEQID NOS: 3 and 4, respectively) Hu9D5VHv4 NCBI accession codes BAC02114and H82, H82b (SEQ ID NO: 10) AAX82494 (SEQ ID NOS: 3 and 4,respectively) Hu9D5VHv4b NCBI accession codes BAC02114 and H47, H108(SEQ ID NO: 11) AAX82494 (SEQ ID NOS: 3 and 4, respectively) Hu9D5VHv5NCBI accession codes BAC02114 and H42, H47, H82b (SEQ ID NO: 12)AAX82494 (SEQ ID NOS: 3 and 4, respectively) Hu9D5VLv1 NCBI accessioncode ABC66952 L36 (SEQ ID NO: 19) (SEQ ID NO: 18) Hu9D5VLv2 NCBIaccession code ABC66952 None (SEQ ID NO: 20) (SEQ ID NO: 18) Hu9D5VLv3NCBI accession code ABC66952 L60 (SEQ ID NO: 21) (SEQ ID NO: 18)Hu9D5VLv4 NCBI accession code ABC66952 L8, L9, L19, L36, L39, L60, L70,L74 (SEQ ID NO: 22) (SEQ ID NO: 18) Hu9D5VLv5 NCBI accession codeABC66952 L8, L9, L18, L19, L36, L39, L60, L70, L74 (SEQ ID NO: 23) (SEQID NO: 18)

TABLE 8 Kabat Numbering of Framework Residues for Backmutations andOther Mutations in Vh Regions of Humanized 9D5 Antibodies BAC02114AAX82494 Mouse Hu9D5 Hu9D5 Hu9D5 Hu9D5 Hu9D5 Hu9D5 Hu9D5 Hu9D5 ResidueHeavy Chain Heavy Chain 9D5 VHv1 VHv2 VHv2b VHv3 VHv3b VHv4 VHv4b VHv5H42 G D E G G E G G G G E H47 W W L L L L W L W L L H69 I I F F F I F II I I H82 M M M S S M S M S M M H82b S S S S L S L S L S L H108 T T T TT L T L T L T

TABLE 9 Kabat Numbering of Framework Residues for Backmutations andOther Mutations in Vk Regions of Humanized 9D5 Antibodies ABC66952 MouseHu9D5 Hu9D5 Hu9D5 Hu9D5 Hu9D5 Residue Light Chain 9D5 VLv1 VLv2 VLv3VLv4 VLv5 L8 P A P P P A A L9 L P L L L P P L18 P S P P P P S L19 A V AA A V V L36 Y F F Y Y F F L39 K R K K K R R L60 D D D D S S S L70 D A DD D A A L74 K R K K K R R

An alignment of the murine 9D5 Vh sequence (SEQ ID NO: 1) with the mousemodel sequences (1SEQ_H and 1MQK_H; SEQ ID NOS: 2 and 62, respectively),the human acceptor sequences (BAC02114 and AAX82494; SEQ ID NOS: 3 and4, respectively), and the Hu9D5VHv1, Hu9D5VHv2, Hu9D5VHv2b, Hu9D5VHv3,Hu9D5VHv3b, Hu9D5VHv4, Hu9D5VHv4b, and Hu9D5VHv5 sequences (SEQ ID NOS:5-12, respectively), is shown in FIG. 1A. The CDRs as defined by Kabatare enclosed in boxes, except that the first enclosed box is a compositeof the Chothia CDR-H1 and the Kabat CDR-H1, with the Kabat CDR-H1underlined and bolded. Positions at which canonical, vernier, orinterface residues differ between mouse and human acceptor sequences arecandidates for substitution. Examples of vernier/CDR foundation residuesinclude residues 2, 49, 69, 71, 75, 80, and 94 by Kabat numbering inFIGS. 12A-E. Examples of canonical/CDR interacting residues includeresidues 24, 48, and 73 by Kabat numbering in FIGS. 12A-E. Examples ofinterface/packing (VH+VL) residues include residues 37, 39, 44, 47, 91,93, and 103 by Kabat numbering in FIGS. 12A-E.

An alignment of the murine 9D5 Vk sequence (SEQ ID NO: 16) with themouse model sequence (1MJU_L; SEQ ID NO: 17), the human acceptorsequence (ABC66952; SEQ ID NO: 18), and the Hu9D5VLv1, Hu9D5VLv2,Hu9D5VLv3, Hu9D5VLv4, and Hu9D5VLv5 sequences (SEQ ID NOS: 19-23,respectively), is shown in FIG. 1B. The CDRs as defined by Kabat areenclosed in boxes, except that the first enclosed box is a composite ofthe Chothia CDR-H1 and the Kabat CDR-H1, with the Kabat CDR-H1underlined and bolded. Positions at which canonical, vernier, orinterface residues differ between mouse and human acceptor sequences arecandidates for substitution. Examples of vernier/CDR foundation residuesinclude residues 4, 35, 46, 49, 66, 68, and 69 by Kabat numbering inFIGS. 13A-D. Examples of canonical/CDR interacting residues includeresidues 2, 48, 64, and 71 by Kabat numbering in FIGS. 13A-D. Examplesof interface/packing (VH+VL) residues include residues 36, 38, 44, 47,87, and 98 by Kabat numbering in FIGS. 13A-D.

The rationales for selection of the positions indicated in Tables 7 and9 in the light chain variable region as candidates for substitution areas follows.

PBA: The model shows a kink in the loop at this position, so abackmutation to A was tried to alleviate this.

L9P: The model shows a kink in the loop at this position, so abackmutation to P was tried to alleviate this.

P18S: P at this position is less frequent. Mutation to S was tried toalleviate loop distortion.

A19V: A and V at this position are equally frequent. Mutation to V wastried to alleviate loop distortion.

Y36F: This is an interface residue, and typically Y or F. Y has an extrahydroxyl group that could potentially affect LC+HC packing. Polarity ofthe Y36 could potentially interfere with placement of heavy chain CRD-H3residue H95. A homology model shows that Y at this position will form ade-novo H-bond with F100(g) in H3 that may result in mobilityrestriction of H3. Both F and Y were used in separate versions.

K39R: R forms H-bonds with adjoining residues in this loop as comparedto K. To rule out any effects on loop stability, mutation to R wastried.

D60S: The presence of D at this residue shows high exposure forproteolysis. In some versions it was replaced with S, a residue mostfrequent in human germ line at this position. This is predicted toenhance stability.

D70A: D has proteolysis potential, so mutation to A was tried.

K74R: R appears to stabilize the loop compared to K at this position, sobackmutation to R was tried.

The rationales for selection of the positions indicated in Tables 7 and8 in the heavy chain variable region as candidates for substitution areas follows.

G42E: E makes ionic interactions with R at position 44. To rule out anyeffect that these interactions might have, backmutation to E was testedin some versions.

W47L: This is an interface residue, typically W. In murine 9D5 heavychain, it is L, whereas in the human accepter framework there is W atthis position. Although L and W are both non-polar, the phenolic ring inW could potentially impact light chain:heavy chain packing. W and L wereincluded in separate versions.

I69F: This is a vernier residue, part of the CDR-H2 foundation.According to the homology model, the aromatic ring of F in murinesequences makes a pie stack with the aromatic ring of CRD-H2 residueY59. An I residue at this position disturbs that stacking. I and F wereincluded in separate versions.

M82S: Mat this position is very rare in human frameworks. More commonare A or N. Changing the residue to the more common S might reduceimmunogenicity that could be associated with the rare M at thisposition. Based upon model observations, there is a possibility that Minteracts with Leu80, which is a vernier zone residue. M and S wereincluded in separate versions.

S82(b)L: S is present at this position in both the murine and humanframework sequences, but S at this position is less frequent. Judgingfrom the position where this residue sits in the model, it possiblycould make contact with the antigen. Residues in framework region 3 ofthe heavy chain are known to occasionally contribute towards binding. Sand L were included in separate versions.

T108L: L is the most frequent residue in human frameworks at thisposition, so L was tried in some versions.

Because two human acceptor frameworks (BAC02114 and AAX82494.1 (SEQ IDNOS: 3 and 4)) were used for humanization of the 9D5 heavy chainvariable region, there are certain framework positions that havedifferent amino acids in the two acceptor frameworks. Humanized versionsof the 9D5 heavy chain variable region can include either amino acid atthese residues. Examples of residues in which these two acceptors differinclude positions H19 (R or K), H40 (A or T), H44 (G or R), H49 (S orA), H77 (S or T), H82a (N or S), H83 (R or K), H84 (A or S), and H89 (Vor M) by Kabat numbering. The rationales for choosing one amino acid orthe other at these positions are as follows.

H19 (R or K): R and K are present in the two frameworks considered, soeach residue was tried in separate versions.

H40 (A or T): A and T are present in the two frameworks considered, soeach residue was tried in separate versions.

H44 (G or R): G and R are present in the two frameworks considered, soeach residue was tried in separate versions.

H49 (S or A): This is a vernier residue that packs under CDR-H2. In themurine sequence, it is A. The slight size difference and hydrophilicnature of S could potentially perturb the CDR foundation. S and A wereincluded in separate versions.

H77 (S or T): S and T are present in the two human acceptor frameworksconsidered, so each was tried in separate versions.

H82a (N or S): N at this position is much less frequent than S.Moreover, S appears to be contributing to antigen binding. Mutation to Swas tried in some versions.

H83 (R or K): K in framework region 3 is close to the CDR bindingsurface area. Replacing it with R, which is bulkier than K, mightobstruct placement of antigen. R is most frequent and K is thirdfrequent in human frameworks at this position. R and K were included inseparate versions.

H84 (A or S): S and A are present in the two frameworks considered, soeach residue was tried in separate versions.

H89 (V or M): V and M are present in the two frameworks considered, soeach was tried in separate versions.

The five humanized light chain variable region variants and fivehumanized heavy chain variable region variants are as follows:

Hu9D5VL version 1 (Y36F substitution in lowercase): (SEQ ID NO: 19)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGNTYLWfLQKPGQSPQLLIYRVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPL TFGQGTKLEIKHu9D5VL version 2 (no substitutions): (SEQ ID NO: 20)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGNTYLWYLQKPGQSPQLLIYRVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPL TFGQGTKLEIKHu9D5VL version 3 (D605 substitution in lowercase): (SEQ ID NO: 21)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGNTYLWYLQKPGQSPQLLIYRVSNLASGVPsRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPL TFGQGTKLEIKHu9D5VL version 4 (P8A, L9P, A19V, Y36F, K39R,D605, D70A, and K74R substitutions in lowercase): (SEQ ID NO: 22)DIVMTQSapSLPVTPGEPvSISCRSSKSLLHSNGNTYLWfLQrPGQSPQLLIYRVSNLASGVPsRFSGSGSGTaFTLrISRVEAEDVGVYYCMQHLEYPL TFGQGTKLEIKHu9D5VL version 5 (P8A, L9P, Pl8S, A19V, Y36F,K39R, D605, D70A, and K74R substitutions in lowercase): (SEQ ID NO: 23)DIVMTQSapSLPVTPGEsvSISCRSSKSLLHSNGNTYLWfLQrPGQSPQLLIYRVSNLASGVPsRFSGSGSGTaFTLrISRVEAEDVGVYYCMQHLEYPL TFGQGTKLEIKHu9D5VH version 1 (W47L, I69F, and M825 substitu- tions in lowercase):(SEQ ID NO: 5) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLElVAEISNSGDTTYYPDTVKGRFTfSRDNAKNSLYLQsNSLKAEDTAVYYCARHYYYGGGYGGWFFDVWGQGTTVTVSSHu9D5VH version 2 (W47L, I69F, M825, and S82(b)Lsubstitutions in lowercase): (SEQ ID NO: 6)EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLElVAEISNSGDTTYYPDTVKGRFTfSRDNAKNSLYLQsNILRAEDTAVYYCARHYYYGGGYGGWFFDVWGQGTTVTVSSHu9D5VH version 2b (G42E, W47L, and T108L substi- tutions in lowercase):(SEQ ID NO: 7) EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYTMSWVRQTPeKRLElVAEISNSGDTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHYYYGGGYGGWFFDVWGQGTlVTVSSHu9D5VH version 3 (I69F, M82S, and S82(b)L substi-tutions in lowercase): (SEQ ID NO: 8)EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVSEISNSGDTTYYPDTVKGRFTfSRDNAKNSLYLQsNlLRAEDTAVYYCARHYYYGGGYGGWFFDVWGQGTTVTVSSHu9D5VH version 3b (W47L and T108L substitutions in lowercase):(SEQ ID NO: 9) EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYTMSWVRQTPGKRLElVAEISNSGDTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHYYYGGGYGGWFFDVWGQGTlVTVSSHu9D5VH version 4 (M825 and S82(b)L substitutions in lowercase):(SEQ ID NO: 10) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVSEISNSGDTTYYPDTVKGRFTISRDNAKNSLYLQsNlLRAEDTAVYYCARHYYYGGGYGGWFFDVWGQGTTVTVSSHu9D5VH version 4b (W47L and T108L substitutions in lowercase):(SEQ ID NO: 11) EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYTMSWVRQAPGKRLElVAEISNSGDTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHYYYGGGYGGWFFDVWGQGTlVTVSSHu9D5VH version 5 (G42E, W47L, and S82(b)L substi-tutions in lowercase): (SEQ ID NO: 12)EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQTPeKRLElVAEISNSGDTTYYPDTVKGRFTISRDNAKNTLYLQMNlLRAEDTAVYYCARHYYYGGGYGGWFFDVWGQGTTVTVSS

Protein quality analysis along with aggregation potential analysesshowed no obvious clusters of aggregation-prone residues present in the9D5 light chain or heavy chain framework regions or CDRs (Wang et al.,mAbs 1(3):254-267, 2009).

Example 8. Binding Kinetic Analysis of Humanized 9D5 Antibodies

Binding kinetics of binding of all humanized 9D5 variants, murine 9D5,and chimeric 9D5 to recombinant human TTR F87M/L110M were characterizedby Biacore, as shown in Table 10. Anti-human (GE Healthcare) wasimmobilized on sensor chip C5 (lacking dextran chains) via aminecoupling following the instructions provided in the GE Healthcareanti-human kit, and mAbs were captured to a level to ensure a maximumbinding of analyte of 30-50 RU. Various concentrations of analyte(recombinant human TTR F87M/L110M) were passed over the captured ligandat 50 ul/min in running buffer (HBS+0.05% P-20, 1 mg/mL BSA) in 3-folddilutions. For each concentration, the reaction proceeded for a timeframe allowing for the higher analyte concentrations to reachequilibrium during association, as well as at least 10% of signal todecay during dissociation. At least one concentration (not the highestor lowest) was run in duplicate. Concentration ranges of analyte wereselected based on preliminary experimentation to span at least 10-foldabove K_(D) to 10-fold below K_(D).

Table 10 summarizes the Biacore association rate (k_(a)), dissociationrate (k_(d)), and binding affinity constant (K_(D)) of the murine,chimeric, and humanized 9D5 variants for recombinant human TTRF87M/L110M.

TABLE 10 Antigen Binding Affinity of 9D5 Antibodies to Human TTR(F87M/L110M) Antibody k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) Rmax Murine 9D52.72E+04 4.93E−04 1.82E−08 31.55 Chimeric 9D5 2.59E+05 3.50E−04 1.35E−0947.05 Hu9D5 H2L5 1.15E+05 7.85E−04 6.84E−09 57.6 Hu9D5 H3L1 1.52E+046.57E−04 4.32E−09 54.93 Hu9D5 H4L1 2.54E+05 5.33E−04 2.09E−09 43.91

These results indicate that the affinity of murine 9D5 forTTR-F87M/L110M (K_(D)=1.82E−08M) has been slightly improved in thechimeric 9D5 variant (K_(D)=1.35E−09M). Furthermore, the fully humanized9D5 variants all have similar affinities in the low nM range. Hu9D5 H4L1has the strongest affinity (K_(D)=2.09E−09) of the humanized 9D5variants tested.

Example 9. Design of Humanized 14G8 Antibodies

The starting point or donor antibody for humanization was the mouseantibody 14G8. The heavy chain variable amino acid sequence of maturem14G8 is provided as SEQ ID NO: 61. The light chain variable amino acidsequence of mature m14G8 is provided as SEQ ID NO: 70. The heavy chainCDR1, CDR2, and CDR3 amino acid sequences are provided as SEQ ID NOS:67-69, respectively (as defined by Kabat). A composite Chothia-KabatCDR-H1 is provided as SEQ ID NO: 118. The light chain CDR1, CDR2, andCDR3 amino acid sequences are provided as SEQ ID NOS: 77-79,respectively (as defined by Kabat). A variant version of CDR1 isprovided as SEQ ID NO: 80. Kabat numbering is used throughout in thisExample.

The variable kappa (Vk) of m14G8 belongs to mouse Kabat subgroup 2,which corresponds to human Kabat subgroup 2. The variable heavy (Vh) ofm14G8 belongs to mouse Kabat subgroup 3d, which corresponds to Kabatsubgroup 1. See Kabat et al. Sequences of Proteins of ImmunologicalInterest, Fifth Edition. NIH Publication No. 91-3242, 1991. The16-residue CDR-L1 belongs to canonical class 3, the 7-residue CDR-L2belongs to canonical class 1, and the 9-residue CDR-L3 belongs tocanonical class 1 in Vk. See Martin & Thornton, J. Mol. Biol.263:800-15, 1996. The 10-residue composite Chothia-Kabat CDR-H1 belongsto canonical class 1, and the 17-residue CDR-H2 belongs to canonicalclass 1. See Martin & Thornton, J Mol. Biol. 263:800-15, 1996. TheCDR-H3 has no canonical classes, but the 15-residue loop probably has akinked base according to the rules of Shirai et al., FEBS Lett.455:188-97 (1999).

The residues at the interface between the Vk and Vh domains are the onescommonly found except L47, at which position the principle amino acid isusually W.

A search was made over the protein sequences in the PDB database(Deshpande et al., Nucleic Acids Res. 33: D233-7, 2005) to findstructures which would provide a rough structural model of 14G8. Thecrystal structure of Fab with esterase activity (pdb 1MJU) was used asthe model for the Vk structure. It was solved at a resolution of 1.22 Aand contains the same canonical structures for CDR-H1 and CDR-H2, andalso contains the same length CDR-H3 with a kinked based. Ananti-cytochrome C oxidase antibody 7E2 Fv fragment (pdb code 1MQK_H) wasused for the Vh structure. It has a resolution of 1.28 A and retains thesame canonical structure for the loops as 14G8. BioLuminate software(licensed from Schrodinger Inc.) was used to model a rough structure of14G8.

A search of the non-redundant protein sequence database from NCBIallowed selection of suitable human frameworks into which to graft themurine CDRs. For Vh, human Ig heavy chains with NCBI accession codesAAD30410.1 and AAX82494.1 were chosen. These share the canonical formsof 14G8 CDR-H1 and H2, and H3 of AAD30410.1 is 15 residues long with apredicted kinked base. For Vk, two human kappa light chains with NCBIaccession codes ABA71374.1 and ABC66952.1 were chosen. They have thesame canonical classes for LCDRs.

Three humanized heavy chain variable region variants and three humanizedlight chain variable region variants were constructed containingdifferent permutations of substitutions (Hu14G8VHv1-3 (SEQ ID NOS:64-66, respectively) and Hu14G8VLv1-3 (SEQ ID NOS: 74-76, respectively))(FIGS. 14A-E and FIGS. 15A-D). The exemplary humanized Vh and Vkdesigns, with backmutations and other mutations based on selected humanframeworks, are shown in FIGS. 14A-E and FIGS. 15A-D, respectively. Thegray-shaded areas in the first column in FIGS. 14A-E and FIGS. 15A-Dindicate the CDRs as defined by Chothia, and the gray-shaded areas inthe remaining columns in FIGS. 14A-E and FIGS. 15A-D indicate the CDRsas defined by Kabat. SEQ ID NOS: 64-66 and 74-76 contain backmutationsand other mutations as shown in Table 11. The amino acids at positionsH1, H3, H47, H105, L8, L9, L19, L26, L36, L60, and L70 in Hui4G8VHv1-3and Hui4G8VLv1-3 are listed in Tables 12 and 13.

TABLE 11 V_(H), V_(L) Backmutations and Other Mutations Donor FrameworkKabat CDR V_(H) or V_(L) Variant V_(H) or V_(L) Exon Acceptor SequenceResidues Residues Hu14G8VHv1 NCBI accession codes AAD30410.1 and H1, H3,H47, H105 — (SEQ ID NO: 64) AAX82494.1 (SEQ ID NOS: 63 and 4,respectively) Hu14G8VHv2 NCBI accession codes AAD30410.1 and H1, H47 —(SEQ ID NO: 65) AAX82494.1 (SEQ ID NOS: 63 and 4, respectively)Hu14G8VHv3 NCBI accession codes AAD30410.1 and H1, H47 — (SEQ ID NO: 66)AAX82494.1 (SEQ ID NOS: 63 and 4, respectively) Hu14G8VLv1 NCBIaccession codes ABA71374.1 and L8, L9, L19, L36, L70 — (SEQ ID NO: 74)ABC66952.1 (SEQ ID NOS: 72 and 73, respectively) Hu14G8VLv2 NCBIaccession codes ABA71374.1 and L36 — (SEQ ID NO: 75) ABC66952.1 (SEQ IDNOS: 72 and 73, respectively) Hu14G8VLv3 NCBI accession codes ABA71374.1and L36, L60 L26 (SEQ ID NO: 76) ABC66952.1 (SEQ ID NOS: 72 and 73,respectively)

TABLE 12 Kabat Numbering of Framework and Kabat CDR Residues forBackmutations and Other Mutations in Humanized 14G8 Antibody VH RegionsAAD30410.1 AAX82494.1 Mouse Hu14G8 Hu14G8 Hu14G8 Residue Heavy ChainHeavy Chain 14G8 VHv1 VHv2 VHv3 H1 Q Q E E E E H3 Q Q K K Q Q H47 W W LL L L H105 Q Q T T Q Q

TABLE 13 Kabat Numbering of Framework and Kabat CDR Residues forBackmutations and Other Mutations in Humanized 14G8 Antibody VL RegionsABA71374.1 ABC66952.1 Mouse Hu14G8 Hu14G8 Hu14G8 Residue Light ChainLight Chain 14G8 VLv1 VLv2 VLv3 L8 P P A A P P L9 L L P P L L L19 A A VV A A L26 S N N N N S L36 Y Y F F F F L60 D D D D D S L70 D D A A D D

An alignment of the murine 14G8 Vh sequence (SEQ ID NO: 61) with themouse model sequence (1MQK_H; SEQ ID NO: 62), the human acceptorsequences (AAD30410.1 and AAX82494.1; SEQ ID NOS: 63 and 4,respectively), and the Hu14G8VHv1, Hu14G8VHv2, and Hu14G8VHv3 sequences(SEQ ID NOS: 64-66, respectively), is shown in FIG. 2A. The CDRs asdefined by Kabat are enclosed in boxes, except that the first enclosedbox is a composite of the Chothia CDR-H1 and the Kabat CDR-H1, with theKabat CDR-H1 underlined and bolded. Positions at which canonical,vernier, or interface residues differ between mouse and human acceptorsequences are candidates for substitution. Examples of vernier/CDRfoundation residues include residues 2, 49, 69, 71, 75, 80, and 94 byKabat numbering in FIGS. 14A-E. Examples of canonical/CDR interactingresidues include residues 24, 48, and 73 by Kabat numbering in FIGS.14A-E. Examples of interface/packing (VH+VL) residues include residues37, 39, 44, 47, 91, 93, and 103 by Kabat numbering in FIGS. 14A-E.

An alignment of the murine 9D5 Vk 14G8 (SEQ ID NO: 70) with the mousemodel sequence (1MJU_L; SEQ ID NO: 71), the human acceptor sequences(ABA71374.1 and ABC66952.1; SEQ ID NOS: 72 and 73, respectively), andthe Hu14G8VLv1, Hu14G8VLv2, Hu14G8VLv3 sequences (SEQ ID NOS: 74-76,respectively), is shown in FIG. 2B. The CDRs as defined by Kabat areenclosed in boxes, except that the first enclosed box is a composite ofthe Chothia CDR-H1 and the Kabat CDR-H1, with the Kabat CDR-H1underlined and bolded. Positions at which canonical, vernier, orinterface residues differ between mouse and human acceptor sequences arecandidates for substitution. Examples of vernier/CDR foundation residuesinclude residues 4, 35, 46, 49, 66, 68, and 69 by Kabat numbering inFIGS. 15A-D. Examples of canonical/CDR interacting residues includeresidues 2, 48, 64, and 71 by Kabat numbering in FIGS. 15A-D. Examplesof interface/packing (VH+VL) residues include residues 36, 38, 44, 47,87, and 98 by Kabat numbering in FIGS. 15A-D.

The rationales for selection of the positions indicated in Tables 11 and12 in the heavy chain variable region as candidates for substitution areas follows.

P8A: Proline is critical to structure conformation, and loss or gain ofproline may affect the structure. Backmutations were designed to avoid“gain of proline.” However, since A at this position is rare in humanIgG frameworks, P was included in some versions.

L9P: Proline is critical to structure conformation, and loss or gain ofproline may affect the structure. Backmutations were designed to avoid“loss of proline.” However, since P at this position is rare in humanIgG frameworks, L was included in some versions.

A19V: A and V have similar frequencies in human frameworks. Bothresidues were included in separate versions.

N26S: L26 represents an N-glycosylation site in light chain CDR1.Mutation to S reduces N-glycosylation and yields a more heterogeneousproduct.

Y36F: This is an interface residue. Substitution was made in someversions to keep the murine residue at this interface residue.

D60S: D and S are similar in frequency in human IgG frameworks. S isapplied by most commercially available therapeutic antibodies, so D wasreplaced with S in some versions.

D70A: D is much more frequent than A in human IgG frameworks. However, Dwill form ionic bond with R24 in light chain CDR1, so both A and D wereincluded in separate versions.

The rationales for selection of the positions indicated in Tables 15 and16 in the heavy chain variable region as candidates for substitution areas follows.

Q1E: Glutamate (E) conversion to pyroglutamate (pE) occurs more slowlythan from glutamine (Q). Because of the loss of a primary amine in theglutamine to pE conversion, antibodies become more acidic. Incompleteconversion produces heterogeneity in the antibody that can be observedas multiple peaks using charge-based analytical methods. Heterogeneitydifferences may indicate a lack of process control.

Q3K: Q is more frequent in human IgG frameworks. K is less frequent butnot rare. Both residues were included in separate versions.

W47L: This is an interface residue. Substitution was made in someversions to keep the murine residue at this interface residue.

Q105T: T may form a hydrogen bond with K3, so T was included in someversions to maintain the conformational structure of VH.

Because two human acceptor frameworks (ABA71374.1 and ABC66952.1 (SEQ IDNOS: 72 and 73, respectively)) were used for humanization of the 14G8light chain variable region, there are certain framework positions thathave different amino acids in the two acceptor frameworks. Humanizedversions of the 14G8 light chain variable region can include eitheramino acid at these residues. An example of a residue in which these twoacceptors differ is position L18 (S or P) by Kabat numbering. Therationale for choosing one amino acid or the other at this position isas follows.

L18 (S or P): P at this position is less frequent. Mutation to S wastried to alleviate loop distortion.

Because two human acceptor frameworks (AAD30410.1 and AAX82494.1 (SEQ IDNOS: 63 and 4, respectively)) were also used as acceptor sequences forhumanization of the 14G8 mature heavy chain variable region, there arecertain framework positions that have different amino acids in the twoacceptor frameworks. Humanized versions of the 14G8 heavy chain variableregion can include either amino acid at these residues. Examples ofresidues in which these two acceptors differ include positions H82a (Nor S), H83 (R or K), H84 (A or S), and H89 (V or M) by Kabat numbering.The rationales for choosing one amino acid or the other at thesepositions are as follows.

H82a (N or S): N and S have similar frequency on 82a. They are alsohuman residues in two human VH templates. S and N were included inseparate versions.

H83 (R or K): K in framework region 3 is close to the CDR bindingsurface area. Replacing it with R might obstruct placement of antigen. Ris most frequent and K is third frequent in human frameworks at thisposition. R and K were included in separate versions.

H84 (A or S): S and A are present in the two frameworks considered, soeach residue was tried in separate versions.

H89 (V or M): V and M are present in the two frameworks considered, soeach was tried in separate versions.

The three humanized light chain variable region variants and threehumanized heavy chain variable region variants are as follows:

Hu14G8VL version 1 (P8A, L9P, A19V, Y36F, D70Asubstitutions in lowercase): (SEQ ID NO: 74)DIVMTQSapSLPVTPGESvSISCRSNKSLLHSNGNTYLWfLQKPGQSPQLLIYRVSNLASGVPDRFSGSGSGTaFTLKISRVEAEDVGVYYCMQHLEYPL TFGQGTKLEIKRHu14G8VL version 2 (L36F substitution in lowercase): (SEQ ID NO: 75)DIVMTQSPLSLPVTPGEPASISCRSNKSLLHSNGNTYLWfLQKPGQSPQLLIYRVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPL TFGQGTKLEIKRHu14G8VL version 3 (N26S, L36F, and D60S substitu- tions in lowercase):(SEQ ID NO: 76) DIVMTQSPLSLPVTPGEPASISCRSsKSLLHSNGNTYLWfLQKPGQSPQLLIYRVSNLASGVPsRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPL TFGQGTKLEIKRHu14G8VH version 1 (Q1E, Q3K, W47L, and Q105T sub-stitutions in lowercase): (SEQ ID NO: 64)eVkLVESGGGLVQPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLElVAEINNSGDTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHYYYGGGYGGWFFDVWGtGTLVTVSSHu14G8VH version 2 (Q1E and W47L substitutions in lowercase):(SEQ ID NO: 65) eVQLVESGGGLVQPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLElVAEINNSGDTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHYYYGGGYGGWFFDVWGQGTLVTVSSHu14G8VH version 3 (Q1E and W47L substitutions in lowercase):(SEQ ID NO: 66) eVQLVESGGGLVQPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLElVAEINNSGDTTYYPDTVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARHYYYGGGYGGWFFDVWGQGTLVTVSS

Example 10. Binding Kinetic Analysis of Humanized 14G8 Antibodies

Binding kinetics of three humanized 14G8 variants and murine 14G8 torecombinant human TTR F87M/L110M were characterized by Biacore, as shownin Table 14. Anti-human (GE Healthcare) was immobilized on sensor chipC5 (lacking dextran chains) via amine coupling following theinstructions provided in the GE Healthcare anti-human kit, and mAbs werecaptured to a level to ensure a maximum binding of analyte of 30-50 RU.Various concentrations of analyte (recombinant human TTR F87M/L110M)were passed over the captured ligand at 50 ul/min in running buffer(HBS+0.05% P-20, 1 mg/mL BSA) in 3-fold dilutions. For eachconcentration, the reaction proceeded for a time frame allowing for thehigher analyte concentrations to reach equilibrium during association,as well as at least 10% of signal to decay during dissociation. At leastone concentration (not the highest or lowest) was run in duplicate.Concentration ranges of analyte were selected based on preliminaryexperimentation to span at least 10-fold above K_(D) to 10-fold belowK_(D).

Table 14 summarizes the association rate (k_(a)), dissociation rate(k_(d)), and binding affinity constant (K_(D)) of the mouse 14G8,chimeric-14G8, Hu14G8 H2L1, Hu14G8 H2L2, Hu14G8H2L3, and Hu14G8 H3L1 forrecombinant human TTR F87M/L110M. As shown in Table 14, Hu14G8antibodies and mouse 14G8 have similar TTR binding affinities.

TABLE 14 Antigen Binding Affinity of 14G8 Antibodies to Human TTR(F87M/L110M) Antibody k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) Rmax Murine14G8 2.88E+04 5.36E−04 1.86E−08 27.13 Chimeric 14G8 2.51E+05 3.46E−041.38E−09 51.06 Hu14G8 H2L1 3.07E+05 6.93E−04 2.26E−09 42.03 Hu14G8 H2L23.26E+05 6.83E−04 2.10E−09 51.43 Hu14G8 H2L3 2.52E+05 3.18E−04 1.24E−0942.41 Hu14G8 H3L1 3.86E+05 6.77E−04 1.76E−09 44.05

Murine 14G8, chimeric 14G8, and other humanized version of 14G8 with nochanges in LCDR1 showed additional light chains when run on SDS-PAGEunder reducing conditions. Sequence analysis revealed that there is anN-glycosylation site in LCDR1 at residue 26 by Kabat numbering. ThisN-glycosylation site may cause potential heterogenecity problems duringmanufacture. Mutation of the N at residue L26 in Hu14G8VLv2 yieldedHu14G8VLv3. Humanized antibodies having Hu14G8VLv3 show only one lightchain when run on SDS-PAGE under reducing conditions, therebyeliminating the potential heterogenecity problem. The antigen bindingaffinity of Hu14G8 H2L3 is similar to that of the murine parent antibodyand the chimeric antibody.

Example 11. Materials and Methods

a. Antibody Generation Protocol

Mice were immunized weekly with the antigenic peptides TTR-MAP,TTR89-97-N-KLH or TTR89-97-C-KLH in RIBI adjuvant or monthly in TiterMaxadjuvant. Three to four days prior to fusion, selected mice were given afinal IV boost with immunogen in saline solution. Spleen werehomogenized to prepare splenocytes and fused with SP2/0 myeloma cellsusing a standard electrofusion protocol. Fused cells in selection mediawere plated in 96-well plates and screened after 7-10 days.

b. Antibody Screening Protocol

Hybridoma selection was based on the following ELISA screen: 96-wellELISA plates were coated with chicken anti-His, 1 μg/mL PBS andincubated for 1 hour. Plates were blocked with of 1% BSA/PBS solution,200 uL/well for 15 minutes then 0.5 μg/mL pH4-TTR, 50 μL/well was addedand incubated for 1 hour. pH4-TTR is TTR that has been subjected to lowpH (50 mM sodium acetate, pH 4.0) in order to dissociate/aggregate TTR,exposing the TTR89-97 epitope. Plates were washed twice with TBS-T.Supernatant from fusion plates was added, 50 μL/well and incubated for 1hour. Plates were washed twice with TBS-T. The detection antibody, goatanti-mouse (IgG1, 2a, 2b, 3 specific)-HRP diluted 1:5,000 in 0.5%BSA/PBS/TBS-T, 50 μL/well was added and incubated for 1 hour. Finally,plates were washed five times with TBS-T and TMB substrate, 100 μL/wellwas added. After 15 minutes, substrate development was stopped with 2NSulfuric Acid, 50 μL/well. Plates were read at 450 nm. Wells with anO.D.>1.0 were selected and cells were transferred to a 24-well plate.After 3 days of growth, clones were counter screened with the aboveassay to confirm binding, and substituting native TTR for pH4-TTR as anegative counter screen, allowing for selection of clones producing TTRmAbs specific for non-native forms of TTR.

c. Antibody Expression Protocols

CMV driven light chain and heavy chain plasmids carrying humanizedmonoclonal antibody sequences were transfected into CHO-S1 cells (LifeTechnology). Dual selection was applied to make a selected pool.Conditioned media was assayed for titer, binding and analyzed bySDS-PAGE/Western blotting. Selected pools were used for clone generationusing Clonepix system (Molecular Devices). Clones were ranked based onantibody titer. Selected clones were expanded and banked.

The highest producing clone was expanded in shake flasks and the culturewas used to inoculate 10-25 L Wave bag cultures. A mixture ofFreeStyle-CHO, CD OptiCHO and FreeStyle F17 expression mediasupplemented with Glutamax (media and Glutamax from Life Technology) wasused for shake flask as well as for Wave bag cultures. Batch culture wasmade using a Wave Bioreactor (GE Healthcare) at 37° C., 7% CO₂ underconstant agitation. Samples were drawn periodically to monitor cellnumber, viability and antibody production. Supplementation with CellBoost (HyClone) was made if needed. The batch culture was harvested whencell viability starts to decline below 90% (5-7 days).

d. Antibody Purification Protocol

The cell culture was harvested after first allowing the cells insuspension to settle down to the bottom of the Wave bag via gravity at4° C. Harvested media was clarified through a depth filter (MillistakPod COHC, Millipore), concentrated 10-fold by tangential flow filtration(Pelicon 2PLC 30K, Millipore) and sterile filtered through a 0.2 μmfilter (Opticap XL, Millipore). The concentrated conditioned media wasthen loaded onto a Protein G Sepharose Fast Flow column (GELifesciences) pre-equilibrated in 1×PBS, pH 7.4 using an FPLC (AktaAvant, GE Lifesciences). Unbound proteins were washed off the columnwith 5-10 column volumes of 1×PBS, pH 7.4 until the OD₂₈₀ reachedbaseline. The bound antibody was eluted from the column with 2 columnvolumes of IgG Elution Buffer (Thermo Scientific). Elution fractionswere collected and pH neutralized with 2M Tris, pH 9.0 (60 μL per 1 mlelution).

Antibody-containing fractions were pooled and dialyzed overnight at 4°C. against 1×PBS, pH 7.4. The dialyzed sample was then sterilized byultrafiltration through a 0.2 μm PES filter and stored at 4° C. Thefinal protein concentration was determined by bicinchoninic acid (BCA)using bovine gamma-globulin as the protein standard (Thermo Scientific).

e. Recombinant TTR Expression and Purification Protocols

E. coli (BL21-A1) cells were transformed with a pET21a(+) plasmidcontaining a TTR insert (Met-hTTR-(His)₆ or a TTR variant containing anF87M/L110M double mutation. Cells were grown in 2YT broth containing 100μg/ml ampicillin. Expression of TTR was induced overnight at 20° C. inthe presence of 1 mM IPTG and 005% arabinose.

The cells were collected by centrifugation at 4000×g for 10 min. andstored at −80° C. until used. 10-15 g cell pellets were thawed and lysedin 50 ml Buffer A (1×PBS containing 500 mM NaCl, 20 mM imidazole) byprocessing through an LV-1 high-shear processor (Microfluidics, Inc.).Lysed cells were centrifuged at 12,000×g for 15 min, filtered through a0.2 μm PES filter prior to purification on a His-Trap HP column (GELifesciences). After loading, the column was washed with 10 c.v. ofBuffer A and eluted with Buffer B (1×PBS with 500 mM NaCl, 500 mMimidazole). Peak fractions corresponding to TTR were collected, dialyzedagainst 1×PBS and stored at −80° C. until used.

f. TTR Antigen Preparation

Native TTR antigen was prepared by diluting a concentrated stock ofrecombinant TTR-6His to a final concentration of 2.5 μg/ml in 1×PBS.pH4-treated TTR was generated by incubating recombinant TTR at aconcentration of 0.2 mg/ml in 50 mM sodium acetate, pH 3.95 for 72 hoursat room temperature. Under these conditions, TTR dissociates intomixture of TTR monomers and aggregated forms that are structurallydistinct from native TTR. The pH4-TTR was then diluted to a finalconcentration of 2.5 μg/ml in 1×PBS immediately before use in the assay.96-well plates (Costar #3690) were coated at room temperature with 50 μlper well of 1.0 μg/ml chicken-anti-his polyclonal antibody (Abcam #Ab9107) in 1×PBS for 1 hr. The coating solution was discarded and theplate was blocked with a 250 μl per well volume of 1×BSA-containingblock buffer diluted in 1×PBS (G-Biosciences #786-193) for 1 hr.

g. ELISA Protocol

Coated and blocked 96-well plates were treated with 50 μl per well of2.5 μg/ml TTR antigen (either native TTR or pH4-TTR) for 1 hr. at roomtemperature. The plates were then washed two times with 250 μl per wellof wash buffer (lx Tris Buffered Saline containing 0.05% Tween-20).Washed plates were then treated with 50 μl per well of the appropriateanti-TTR monoclonal antibody at concentrations ranging from of 0.31 to2.5 μg/ml, for 1 hr.

The treated plates were washed 3 times with 250 μl per well wash buffer.After washing, the plates were treated for 1 hr. with 50 μl per well ofdetection antibody comprising a 1:5,000 dilution of peroxide-conjugatedgoat-anti-mouse (Jackson ImmunoResearch #115-035-164) in 1×PBS. Theplate was then washed 3 times prior to the addition of 100 μl per wellTMB substrate (Rockland). The HRP reaction was allowed to proceed atroom temperature for 15 min. before quenching with a 50 μl per wellvolume of 1N H₂SO₄. Spectroscopic absorbance was measured at awavelength of 450 nm.

h. SDS-PAGE

Electrophoresis on SDS-polyacrylamide gels was carried out as follows.0.1-1 μg TTR or pH 4.0-TTR in 1×LDS sample buffer (Life Technologies)was loaded onto a 10% NuPAGE bis-tris gel and subjected toelectrophoresis in MES buffer at a constant 90V for 105 minutes. Afterelectrophoresis, the gel was either stained in Instant Blue (Expedeon)or transferred to nitrocellulose filters for Western blot analysis.

i. Native PAGE

Electrophoresis on native Tris-glycine gels was carried out as follows.0.1-1 μg TTR or pH 4.0-TTR in 1×Tris-glycine sample buffer (LifeTechnologies) was loaded onto a 10-20% Tris-glycine gel and subjected toelectrophoresis in 1× Native Tris-glycine running buffer at a constant120V for 105 minutes. After electrophoresis, the gel was either stainedin Instant Blue (Expedeon) or transferred to nitrocellulose filters forWestern blot analysis.

j. Western Blot

SDS- or Native-PAGE gels were blotted onto nitrocellulose filter paper(iBlot, P7 Program) and blocked with blocking buffer (Licor) for 30minutes. The filters were then incubated in 0.5 μg/ml primary antibodyin blocking buffer for 1 hour at room temperature (or over-night at 4°C.), followed by three, 10 minutes washes with 1×TBS. The filters wereplaced in IRDye 800CW-conjugated goat-anti-mouse secondary diluted1:20,000 in block buffer. After incubating the filters in secondaryantibody solution for 1 hour at room temperature, the filters werewashed and imaged on an Odyssey CLx infrared imager (Licor).

k. TTR Fiber Formation Assay Procotol

A solution of 3.6 μM (0.2 mg/ml) TTR-Y78F in 50 mM sodium acetate, pH4.8 was incubated at 37° C. for 72 hours in the presence of 1.4 μM (0.2mg/ml) mis-TTR antibody or an isotype control. After incubation, a 5×molar excess of thioflavin-T was added to the mixture and allowed tobind for 30 minutes. Fluorometric measurements were measured at anemissions wavelength of 480 nm with an excitation wavelength set at 440nm. The 0% inhibition was set as the fluorescence intensity in thepresence of an isotype control antibody (83 a.u.) and the 100%inhibition point was set as the fluorescence in the absence of TTR-Y78Fprotein (38 a.u.).

A stock solution of TTR-V122I (approximately 5 mg/mL) was diluted intobuffer with final concentrations of 0.2 mg/mL TTR, 30 mM sodium acetate,5 mM sodium phosphate, 100 mM KCl, 1 mM EDTA, 0.02% sodium azide, andvarying concentrations of monoclonal antibody at pH 7.2. This sample wasdialyzed against the same buffer at pH 4.5 for 3 h at room temperature.Samples were then incubated at 37° C. for 72 hours. After incubation a5× molar excess of thioflavin-T was added to was added to the mixtureand allowed to bind for 30 minutes. Thioflavin-T fluorescence wasmonitored using a Photon Technology International C60spectrofluorimeter. The photomultiplier gain was varied and excitationand emission slit widths set to 2-4 nm to maximize signal to noise.Fluorescence measurements were made using 430 nm and 480 nm asexcitation and emission wavelengths, respectively.

l. Cardiac Tissue Samples

Fresh frozen and paraffin-processed blocks of cardiac tissue withconfirmed diagnoses of ATTR mutations were obtained from Dr. MerrillBenson at Indiana University. Samples included eight fresh frozensamples and six FFPE samples and each sample was diagnosed with eitherATTR or some other cardiac amyloidosis. The diagnosis of the tissue wasfurther confirmed at Prothena via IHC staining with antibodies to kappaand lambda light chains and amyloid A prior to characterization with theTTR antibodies.

m. Immunohistochemistry

Immunohistochemistry was performed on lightly paraformaldheyde-fixed, 10μm slide-mounted cryosections and on 5 μm paraffin sections. Theimmunoperoxidase method was the principal detection system, which wasperformed on the Leica Bond Rx (Leica Biosystems, Buffalo Grove, Ill.)using the Bond Polymer Refine Detection Kit (DS980, Leica Biosystems).The primary antibodies were incubated for one hour (according toconcentrations in Table 2) followed by incubation with anti-mouse andanti-rabbit polymeric HRP-linker antibody conjugates. The staining wasvisualized with a DAB chromogen, which produced a brown deposit. Theslides were counterstained with hematoxylin, dehydrated in an ascendingseries of alcohols, cleared in xylenes, and coverslipped with CytoSeal60 (Richard Allen Scientific; Kalamazoo, Mich.). Negative controlconsisted of performing the entire immunohistochemical procedure onadjacent sections with a non-immune IgG isotype control or an omissionof the primary antibody.

n. Demonstration of Amyloid: Congo Red and Thioflavin T Staining

Congo red stain was performed to demonstrate TTR amyloid in the tissueusing a kit from American MasterTech (Lodi, Calif.). The staining wasperformed according to the manufacturer's procedure. Slides were stainedin the Congo Red solution for 1 hour followed by differentiation in 1%sodium hydroxide for approximately 15 seconds. The slides were thenrinsed in running water, dehydrated through an alcohol series ofincreasing concentrations, and cleared through three changes of xylenes,and coverslipped with CytoSeal 60.

A modified Thioflavin T staining protocol (Schmidt et al 1995.) wasemployed to determine the presence of TTR amyloid in the tissue.Briefly, slides were counterstained with a Mayers hematoxylin, rinsed inrunning water and stained with a filtered solution of 0.015% ThioflavinT (T3516-25G; Sigma-Aldrich, St. Louis, Mo.) in 50% ethanol for tenminutes. The slides were then rinsed in running water and differentiatedin 1% (v/v) acetic acid for 10 minutes and rinsed three times in water.The slides were allowed to air dry before being coverslipped withProLong Gold (Life Technologies).

o. Image Analysis

Slides were imaged with either an Olympus BX61 microscope, HamamatsuNanozoomer 2.0HT digital slide scanner, or a Leica SPE spectral confocalsystem. Images were collected and stored as TIFF files.

p. Analysis of Human Plasma Samples by SDS-PAGE/Western

Six plasma samples from patients confirmed for V30M ATTR (Sample #11,#12, #15, #18, #19, #20) and 6 samples (#21, #22, #23, #24, #25, #27)from normal subjects were obtained from M. Saraiva (Porto University,Portugal). Sample # C6 was a normal human serum sample obtained from acommercial source (BioreclamationIVT). These plasma samples wereseparated by SDS-PAGE and Western blotted with 9D5 or 5A1 as follows. A1.4 μl volume of plasma was diluted 1:8 into 1×LDS sample buffer in theabsence of reducing agent (Life Technologies). Samples were subjected toSDS-PAGE separation and Western blotted with 0.5 μg/ml 9D5 or 5A1 asdescribed previously.

q. Analysis of Human Plasma Samples by MesoScale Discovery (MSD) PlateAssay

96-well MSD plates were coated with monoclonal antibody 6C1 at aconcentration of 4 μg/mL in PBS and incubated for 2 hours at roomtemperature with shaking, or overnight at 4° C. Plates were washed threetimes with 1×TBST before being blocked with of 3% MSD Blocker Asolution, 150 μL per well for 1 hour shaking. A 30 μl per well volume ofhuman plasma samples diluted 1:10 in a sample buffer comprised of 0.6%globulin-free bovine serum albumin, 1.5 mM monobasic sodium phosphate, 8mM dibasic sodium phosphate, 145 mM sodium chloride, 0.05% Triton X-405,and 0.05% thimerosal was added to the blocked MSD plates for 1 hour.Plates were washed 3 times with 1×TBST. A 50 μl per well volume of 1μg/ml sulfo-tagged detection antibody (either 8C3 total TTR antibody ofthe Dako polyclonal antibody) in sample buffer was added for 1 hr. atroom temperature with shaking. Plates were washed three times with1×TBST followed by the addition of 150 μl per well 1× Read Buffer Tsolution (Meso Scale Discovery). Plates were then read in the MSD Sectorimager.

r. Generation of an MSD Standard Curve

In order to quantitate the amount of non-native, 6C1-reactive TTRprotein present in human plasma samples, a MSD standard curve wasgenerated using recombinant TTR-F87M/L110M as a 6C1-reactive TTRstandard. This TTR variant contains two amino acid substitutions thatprevent tetramer formation and keeps the protein in the monomer state(Jiang et al. (2001) Biochemistry 40, 11442-11452). As such, this TTRvariant is recognized by all mis-TTR mAbs and is therefore well-suitedfor use as a reference standard in the MSD assay.

To generate the standard curve, 96-well MSD plates were coated withmis-TTR antibody 6C1 at a concentration of 4 μg/mL in PBS and incubatedfor 2 hours at room temperature with shaking, or overnight at 4° C.Plates were washed three times with 1×TBST before being blocked with of3% MSD Blocker A solution, 150 μL per well for 1 hour shaking. Theblocked plates were then treated for 1 hour with 50 μl per well of 25μg/mL TTR-F87M/L110M serially diluted 1:5 with the last dilution being abuffer blank. Plates were washed 3 times with 1×TBST before the additionof a 50 μl per well volume of 1 μg/ml SulfoTag-detection antibody(8C3-SulfoTag or Dako pAb-SulfoTag) for 1 hour at room temperature withshaking. Both 8C3 mAb and the Dako antibody were coupled to the SulfoTagand could be used at the detection antibody since they bound to totalTTR and were not conformation specific.

After treatment with the detection antibody, plates were washed threetimes with a 150 μl per well volume of 1×TBST, followed by the additionof 150 μl per well 1× Read Buffer T (MSD). Plates were read in the MSDSector imager and a resulting TTR F87M/L110M calibration curve wasgenerated.

s. Phagoctyosis Assay

A 1-mg/mL sample of TTR-F87M/L110M, native TTR or low-pH aggregatedTTR-V30M was amine coupled with pHrodo dye for 15 min at 37° C. with aprotein:dye ratio of ˜15:4 according to the manufacturer'sspecifications (Thermo Scientific). Excess pHrodo-label was removed bydiafiltration in a spin concentrator with a 10K molecular weight cutoff(Pierce Thermo) and the pHrodo-TTR was resuspended in 1×PBS.

THP-1 human monocytes were cultured in cell culture media (RMPI, 10% lowIgG serum, pen/strep). A 20-μg/mL aliquot of pHrodo-labeled TTR wasseparately pre-incubated with 40 μg/mL antibody at 37° C. in cellculture media for 30 min prior to the addition of 5E+04 THP-1 cells in a1:1 volumetric ratio. After tissue culture incubation (3 h), cells werewashed with cell culture media three times, incubated in media for 10min, then washed twice with and resuspended in FACS buffer (1% FBS inPBS). Red pHrodo fluorescence intensity was detected using Texas Redchannel filters. Epifluorescence microscopy was carried out in a similarfashion. After FACS analysis, the remaining cells were transferred toglass chamber slides and imaged by inverted microscopy. Meanfluorescence intensities were automatically calculated by averaging therelative fluorescence intensities of each individual cell.

Example 12. Antibody-Dependent Uptake of TTR by THP-1 Cells

TTR-F87M/L110M was covalently labeled with the pH-sensitive fluorescentdye pHrodo. The pHrodo tag has minimal fluorescence under physiologicalpH, but fluorescence is enhanced upon engulfment into the low pHenvironments of endocytic vesicles and thus marks cellular uptake oftagged particles. THP-1 monocytes were added to pHrodo-tagged TTR(native or non-native, TTR-F87M/L110M) after treatment with either 14G8or the isotype control antibody. Low levels of fluorescence wereobserved with either antibody incubated with native TTR, and afterincubation of the control antibody with non-native TTR. Fluorescence wasincreased, however, after 14G8 incubation with non-native TTR (FIG. 7A),suggesting that non-native TTR is not efficiently phagocytized underbasal conditions, however the addition of mis-TTR antibodiesspecifically elicits phagocytosis of non-native TTR.

Dose-dependent phagocytosis of pHrodo-labeled, large aggregatedfibrillar particles of TTR was also demonstrated for each of the mis-TTRmAbs (FIG. 7B). Maximum antibody-dependent uptake was variable for eachmis-TTR mAb (6C1>9D5≈14G8>5A1), reaching a plateau at mAb concentrationsbetween 5-10 μg/mL. Variable antibody potencies may reflect isotypedifferences and associated changes in effector function among the fourmis-TTR mAbs. Controls, including untreated cells or those treated withan IgG1 isotype control, did not demonstrate detectable or enhancedfluorescence, respectively.

Example 13. Evaluation of mis-TTR Antibodies in Transgenic Mouse Model

In vivo studies are conducted in a humanized transgenic mouse model V30MhTTR (Inoue et al., (2008) Specific pathogen free conditions preventtransthyretin amyloidosis in mouse models. Transgenic Research17:817-826) to assess the efficacy of anti-TTR antibodies in the bindingand removal of aggregated hTTR.

Transgenic mice are bred using standard procedures and their circulatinghTTR levels are assessed by ELISA. Mice with a serum level of 200-400μg/ml of hTTR are used for subsequent efficacy studies. The first set ofstudies examine the natural deposition of hTTR in transgenic mice.Detection of hTTR deposits begins at 12 months of age and is repeatedevery 3-6 months thereafter. Once an acceptable level of aggregates isseen in transgenic mice, efficacy studies are initiated. Animals aredivided into three treatment groups (n=10/group) and treated weekly forfour weeks with an IP dose of vehicle, control antibody (isotypecontrol, 10 mpk) or an anti-hTRR antibody (10 mpk). One week after thelast treatment the mice are euthanized, tissues collected and processed,and then stained to assess the number and size of remaining TTRdeposits. Quantitative methods and statistics are employed to determinethe degree of clearance seen among groups.

In an alternative approach, hTTR aggregates are prepared in vitro andthen injected into the kidney of transgenic mice to seed the depositionof new aggregates. Applicant has determined that the injection of thesepreparations can expedite the deposition of new aggregates in apredictable manner. Based on these findings, animals are sedated, theleft kidney exposed and pre-aggregated hTTR mateiial injected into thecortex of the kidney. After a suitable recovery period, mice are dividedinto three treatment groups (n=10/group) and treated weekly forfour-eight weeks with an IP dose of vehicle, control antibody (isotypecontrol, 10 mpk) or an anti-hTRR antibody (10 mpk). One week after thelast treatment the mice are euthanized, the kidneys collected andprocessed, and then stained to assess the number and size of TTRdeposits. Quantitative methods and statistics are employed to determinethe degree of change seen among groups.

Example 14. Evaluation of mis-TTR Antibodies in a Matrigel implant Model

Applicant has determined that pre-aggregated hTTR can be suspended inMatrigel (BD Bioscience, Cat #354263), allowed to solidify and thenplaced subcutaneously in mice. At four weeks post implantation, theMatrigel implant maintained its structure and the aggregated hTTR wasstill present within the implant. Moreover, the implant was welltolerated by the mice and anti-hTTR antibodies were able to penetrateand hind to the aggregates suspended in the Matrigel. Based on thesefindings, an antibody efficacy study is conducted. Animals are sedatedand an implant containing pre-aggregated hTTR suspended in Matrigelplaced subcutaneously in mice. After a suitable recovery period, miceare divided into three treatment groups (n=10/group) and treated weekly,for two-four weeks with an IP dose of vehicle, control antibody (isotypecontrol, 10 mpk) or an anti-hTRR antibody (10 mpk). After the lasttreatment, the mice are euthanized, the skin containing the implantcollected and processed, and then the amount of TTR deposits remainingassessed using histological and/or biochemical methods. Quantitativeanalysis and statistics are employed to determine the degree ofclearance seen among groups.

Example 15. Electron and Atomic Force Microscopy

Immunogold transmission electron microscopy (TEM) and atomic forcemicroscopy (AFM) were used to generate images of the interaction betweenmis-TTR mAbs and both aggregated and fibrillar forms of the protein.Isothermal titration calorimetry (ITC) was carried out by titrating 14G8into a solution of aggregated TTR using standard methods. Using TEM,immunogold labeling with 14G8 was observed in TTR-V122I oligomeraggregates and fibril ends (FIG. 8A), whereas immunogold labeling withan anti-TTR pAb shows binding along the lengths of TTR fibers and tooligomeric clusters (FIG. 8B). IgG1 isotype control mAb does not showimmunogold labeling (FIG. 8C). TTR-V122I fibers, alone and in thepresence of 14G8±6 nm colloidal gold-conjugated secondary antibody, wereassessed using AFM. Gold labeling was observed at fiber ends (FIG. 8D).FIG. 9A shows isothermal titration calorimetry (ITC) data and bindingisotherms for 14G8 binding to aggregated TTR variants. FIG. 9B showsbinding fitted to a 2-binding site model with KD values shown. TEM, AFM,and ITC analysis provide evidence mis-TTR mAbs bind to TTR aggregatesand fibrils primarily at 2 distinct sites: oligomers and fibril ends.

Example 16. Antibody Binding to TTR Amyloid in the Peripheral Nerves andGastrointestinal Tract of a Patient with ATTR Amyloidosis from aTTR-V30M Mutation

14G8 and control antibodies were evaluated immunohistochemically todetermine their reactivity for TTR amyloid deposits in nerve, andgastrointestinal tract samples obtained from patients with confirmeddiagnoses of ATTR amyloidosis from a V30M mutation. FIGS. 10A-G show14G8 immunolabeled TTR amyloid present between fibers of the nervefascicle (FIG. 10A panels 1 and 2), which overlapped with staining byCongo red (FIG. 10B panels 1 and 2) and thioflavin T (FIG. 10C panels 1and 2), and immunolabeling by a total-TTR antibody (FIG. 10D) in tissuederived from a patient with ATTR amyloidosis. No staining was seen withthe use of 2 isotype control antibodies (FIGS. 10E-F); however, axonaldegeneration (lack of Schwann cell nuclei) in the areas laden with TTRamyloid deposits were also observed (FIGS. 10E-F [red areas in 10E]).Peripheral nerves from a healthy control were not labeled using either14G8 or a total-TTR antibody (FIG. 10G panels 1-3).

14G8 labelled TTR amyloid deposited throughout the gastrointestinaltract; Meissner's plexus and glands in the esophagus (FIGS. 11A, Bpanels 1), the rich vasculature bed in the submucosa (FIG. 11C panel 1),and the muscularis propria (MP) and muscularis mucosa (MM) of thejejunum (FIG. 11D panel 1) were immunolabeled with 14G8. 14G8-positiveTTR amyloid overlapped with Congo red fluorescent staining (FIGS. 11A-Dpanels 2). ATTR amyloidosis tissue stained with an isotype control mAb(FIGS. 119A-D panels 3). 14G8 immunoreactivity was absent in healthycontrol tissue (FIG. 11E panels 1-4).

These findings provide evidence that mis-TTR mAbs can be useful inpreventing the deposition or enhancing the clearance, or both, of TTRamyloid in patients with ATTR amyloidosis regardless of the specificorgan(s) involved while sparing the function of the normal tetramericform of the protein.

What is claimed is:
 1. An antibody that binds to transthyretin andcomprises three Kabat heavy chain CDRs of SEQ ID NOS: 13-15 respectivelyand three Kabat light chain CDRs of SEQ ID NOS: 24-26 respectively. 2.The antibody of claim 1, wherein CDR-H1 is a composite Kabat-Chothia CDRof SEQ ID NO:
 117. 3. The antibody of claim 1 that is a chimeric,humanized or veneered antibody.
 4. The antibody of claim 1 that has ahuman IgG1 isotype.
 5. The antibody of claim 1 that has a human IgG2 orIgG4 isotype.
 6. The antibody of claim 3, which is humanized, andcomprises a humanized mature heavy chain variable region having an aminoacid sequence at least 90% identical to any one of SEQ ID NOS: 5-12 anda humanized mature light chain variable region having an amino acidsequence at least 90% identical to any one of SEQ ID NOS: 19-23,provided that position H19 is R or K, position H40 is A or T, positionH44 is G or R, position H49 is S or A, position H77 is S or T, positionH82a is N or S, position H83 is R or K, position H84 is A or S, andposition H89 is V or M.
 7. The humanized antibody of claim 6, whereinpositions H47 and H108 are each occupied by L and position L36 isoccupied by F.
 8. The antibody of claim 1, wherein the mature heavychain variable region has the amino acid sequence of any one of SEQ IDNO: 5-12 and the mature light chain variable region has the amino acidsequence of any one of SEQ ID NO: 19-23.
 9. The antibody of claim 8,wherein the mature heavy chain variable region has the amino acidsequence of SEQ ID NO: 11 and the mature light chain variable region hasthe amino acid sequence of SEQ ID NO:
 19. 10. The antibody of claim 1that is an intact antibody.
 11. The antibody of claim 1 that is abinding fragment.
 12. The antibody of claim 11, wherein the bindingfragment is a single-chain antibody, Fab, or Fab′2 fragment.
 13. Theantibody of claim 11, which is humanized, and wherein the mature lightchain variable region is fused to a light chain constant region and themature heavy chain variable region is fused to a heavy chain constantregion.
 14. The humanized antibody of claim 13, wherein the heavy chainconstant region is a mutant form of a natural human heavy chain constantregion which has reduced binding to a Fcγ receptor relative to thenatural human heavy chain constant region.
 15. The humanized antibody ofclaim 13, wherein the mature heavy chain variable region is fused to aheavy chain constant region having the sequence of SEQ ID NO: 103 withor without the C-terminal lysine and/or the mature light chain variableregion is fused to a light chain constant region having the sequence ofSEQ ID NO: 104 or
 105. 16. A pharmaceutical composition comprising theantibody of claim 1 and a pharmaceutically acceptable carrier.
 17. Anucleic acid or nucleic acids encoding the heavy chain and light chainof an antibody as described in claim
 1. 18. The nucleic acid or nucleicacids of claim 17 having a sequence comprising any one of SEQ ID NOS:40, and 44-50 encoding a heavy chain variable region, any one of SEQ IDNOS: 42 and 51-56 encoding a light chain variable region, SEQ ID NO: 106encoding a heavy chain constant region and SEQ ID NO: 107 or 108encoding a light chain constant region.
 19. A recombinant expressionvector comprising a nucleic acid or nucleic acids of claim
 17. 20. Ahost cell transformed with the recombinant expression vector of claim19.
 21. A method of humanizing an antibody, the method comprising: (a)selecting one or more acceptor antibodies; (b) identifying the aminoacid residues of the mouse antibody to be retained; (c) synthesizing anucleic acid encoding a humanized heavy chain comprising CDRs of themouse antibody heavy chain and a nucleic acid encoding a humanized lightchain comprising CDRs of the mouse antibody light chain; and (d)expressing the nucleic acids in a host cell to produce a humanizedantibody; wherein the mouse antibody is characterized by a mature heavychain variable region of SEQ ID NO: 1 and a mature light chain variableregion of SEQ ID NO:
 16. 22. A method of producing a humanized,chimeric, or veneered antibody, the method comprising: (a) culturingcells transformed with nucleic acids encoding the heavy and light chainsof the antibody, so that the cells secrete the antibody; and (b)purifying the antibody from cell culture media; wherein the antibody isa humanized, chimeric, or veneered form of an antibody characterized bya mature heavy chain variable region of SEQ ID NO: 1 and a mature lightchain variable region of SEQ ID NO:
 16. 23. A method of producing a cellline producing a humanized, chimeric, or veneered antibody, the methodcomprising: (a) introducing a vector encoding heavy and light chains ofan antibody and a selectable marker into cells; (b) propagating thecells under conditions to select for cells having increased copy numberof the vector; (c) isolating single cells from the selected cells; and(d) banking cells cloned from a single cell selected based on yield ofantibody; wherein the antibody is a humanized, chimeric, or veneeredform of an antibody characterized by a mature heavy chain variableregion of SEQ ID NO: 1 and a mature light chain variable region of SEQID NO:
 16. 24. A method of inhibiting or reducing aggregation oftransthyretin in a subject having a transthyretin-mediated amyloidosis,comprising administering to the subject an effective regime of theantibody of claim 1 thereby inhibiting or reducing aggregation oftransthyretin in the subject.
 25. A method of inhibiting or reducingtransthyretin fibril formation in a subject having atransthyretin-mediated amyloidosis, comprising administering to thesubject an effective regime of the antibody of claim 1, therebyinhibiting or reducing transthyretin accumulation in the subject.
 26. Amethod of reducing transthyretin deposits in a subject having atransthyretin-mediated amyloidosis, comprising administering to thesubject an effective regime of the antibody of claim 1, thereby reducingtransthyretin deposits in the subject.
 27. A method of clearingaggregated transthyretin in a subject having a transthyretin-mediatedamyloidosis, comprising administering to the subject an effective regimeof the antibody of claim 1, thereby clearing aggregated transthyretinfrom the subject relative to a subject having a transthyretin-mediatedamyloidosis who has not received the antibody.
 28. A method ofstabilizing a non-toxic conformation of transthyretin in a subjecthaving a transthyretin-mediated amyloidosis, comprising administering tothe subject an effective regime of the antibody of claim 1, therebystabilizing a non-toxic conformation of transthyretin in the subject.29. A method of treating a transthyretin-mediated amyloidosis in asubject, comprising administering to the subject an effective regime ofthe antibody of claim
 1. 30. A method of delaying the-onset or lesseningthe severity of a transthyretin-mediated amyloidosis in a subject,comprising administering to the subject an effective regime of theantibody of claim
 1. 31. The method of claim 24, wherein thetransthyretin-mediated amyloidosis is selected from cardiomyopathy,hypertrophy, familial amyloid polyneuropathy, central nervous systemselective amyloidosis (CNSA), senile systemic amyloidosis, senilecardiac amyloidosis, spinal stenosis, osteoarthritis, rheumatoidarthritis, juvenile idiopathic arthritis, age related maculardegeneration, and a ligament or tendon disorder.
 32. A method ofdiagnosing a transthyretin-mediated amyloidosis in a subject, comprisingcontacting a biological sample from the subject with an effective amountof the antibody of claim 1.